Polyphosphate Metabolism in Micro-Organisms

Polyphosphate Metabolism in Micro-Organisms IGOR S. KULAEV and VLADIMIR M. VAGABOV Institute of Biochemistry and Physiology of Micro-organisms, Academy of Sciences of the U.S.S.R., Pushchino, Moscow Region, U.S.S.R. I Introduction

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A. Inorganic polyphosphates

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B. Distribution in micro-organisms . . C. Methods of detection, identification and fractionation of inorganicpolyphosphates . . . . . . . . . . . . 11. High molecular-weightpolyphosphates . . . . . . A. Intracellular localization . . . . . . . . . B. Enzymes involved in biosynthesis and degradation of polyphosphates . C. Metabolism of polyphosphates in eukaryotes . . . . . . D. New data on polyphosphate metabolism in prokaryotes . . . . E. Concluding remarks on the physiological role of high molecular-weight . . . . polyphosphates in microbial metabolism 111. Inorganic pyrophosphate: new aspects of metabolism and physiological role . A. Utilization of pyrophosphate in phosphorylation reactions in bacteria . B. Energy-dependent synthesis of pyrophosphate during photosynthetic and . . . . . . . . oxidative phosphorylation . C. Relationship between pyrophosphate and polyphosphate metabolism in . . . . . . . . . . micro-organisms IV. Modem concepts about the role of high molecular-weightpolyphosphates and . . . . pyrophosphate in evolution of phosphorous metabolism V. General conclusions . . . . . . . . . . VI. Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . .

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83 84 85

86 89 89 103 114 132 141 142 142 145 150 153 157 158 158

I. Introduction One of the topical problems of modern biochemistry is the elucidation of the mechanisms underlying the synthesis and utilization of energy-rich phosADVANCES IN MICROBIAL PHYSIOLOGY, ISBN 0-12027724-7 VOL. 24

Copyright 0 1983 Academic Press London All rights of reproduclion in my form reserved.

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phates, among which the major role is obviously played by nucleoside di- and triphosphates, and in the first place by the ATP-ADP system. However, numerous findings reported in the last three decades show that cell energetics are not inconsiderably contributed to by compounds other than nucleoside polyphosphates. These include so-called condensed inorganic phosphates which are found primarily in micro-organisms and appear to be primitive energy accumulators in living organisms (Belozersky, 1959). They were first reported at the end of the last century (Liebermann, 1888); however, a thorough study of their structure and metabolism began only in the middle of this century, when the investigations carried out mainly by Jeener and Brachet (1 944), Wiame (1949), Ebel (1951), Belozersky ( 1958), Kulaev and Belozersky (1962), Lohmann and Langen (1956), HoffmannOstenhof and Weigert (1952), Hoffmann-Ostenhof (1962), Kornberg et al. (1956), Harold (1966) and some others (see Dawes and Senior, 1973; Kulaev, 1973a,b, 1979) laid the foundations of the biochemistry of inorganic polyphosphates. Ever-increasingattention has in recent years been paid to new aspects of the biochemistry of these extremely interesting phosphorus-containing compounds. Despite the availability of a number of reviews pertaining to this field of knowledge (Kuhl, 1960, 1974; Harold, 1966; Dawes and Senior, 1973; Kulaev, 1975) and the publication of a monograph on the biochemistry of high molecular-weight polyphosphates (Kulaev, 1979), it seems necessary today to revise certain concepts of the basic features of the metabolism and physiological role of these high-energy phosphorus compounds. Many problems of their biochemistry, extensively reviewed in the abovementioned publications, will not be discussed here. In this review, accent will be laid on the problems the study of which has recently yielded principally new data, and on the aspects which have not been discussed earlier in sufficient detail.

A . INORGANIC POLYPHOSPHATES

Inorganic polyphosphates are linear polymers in which orthophosphate residues are linked by energy-rich phospho-anhydride bonds (Yoshida, 1955; Flodgaard and Fleron, 1974; Fig. 1). The number of phosphate residues in these compounds, as identified in living organisms, may vary noticeably: from two in the simplest compound of this type, pyrophosphate, to several hundreds and thousands in high molecular-weight polyphosphates (Kulaev, 1979). The structure and properties of polyphosphates are described in a number of reviews and monographs (Van Wazer, 1958; Boulle, 1965; Ohashi, 1975; Kulaev, 1979).

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

r

85

1( n + 2 ) -

FIG. 1. Molecular structure of a linear polyphosphate. Me’ is a monovalent metal. From Thilo (1959).

B. DISTRIBUTION IN MICRO-ORGANISMS

Inorganic polyphosphates have been found in almost all tested representatives of living cells (Kulaev, 1979). They have been detected in eubacteria, fungi, algae, mosses, protozoa, insects, and in various tissues of higher plants and animals. Unfortunately, up to date no attempts have been made to detect these compounds in representatives of a new realm of living beings, namely the archaebacteria (woese and Fox, 1977; Steckenbrandt and Woese, 1979). The quantities of high molecular-weight polyphosphates detected hitherto in cells of higher plants and animals are small. According to published data, their phosphorus contents amount to tens, at most hundreds, of micrograms per gram wet tissue of these organisms. As to cells of micro-organisms, the situation is just the opposite. Yeast, for example, when grown in a medium with phosphate and glucose and certain cations (K+, Mg*+), after phosphorus starvation, may accumulate polyphosphates in amounts of up to 20% of the cell dry weight. Liss and Langen (1960) called this phenomenon “Polyphosphat ~berkompensation”which has been translated into English as “polyphosphate overplus” (Harold, 1964). Such a “polyphosphate overplus” occurs in cells in the absence of growth, i.e. when most of the energy has been released during glucose oxidation and the bulk of phosphate absorbed accumulated in polyphosphates. However, some intensively growing bacteria, e.g. Acinetobacter, during cultivation on butyrate, are capable of accumulating, besides a substantial amount of lipids, inorganic polyphosphates in quantities of 10-20% of the dry weight (Deinema et af.,1980). It is noteworthy that, in this case, uptake of a large amount of exogenous phosphate and its accumulation inside the cells in the form of polyphosphate granules are characteristic of normal metabolism of these bacteria. In contrast to the “polyphosphate overplus”, this phenomenon has been termed “luxury uptake” (Levin and Shapiro, 1965).

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Metabolism of polyphosphates in microbial cells has been studied in most detail since, in micro-organisms, these compounds accumulate in significant quantities. Nevertheless, inorganic polyphosphates are not vitally important for living organisms, and do not appear to be obligatory cell components. This has been demonstrated by Harold, who obtained mutants of Aerobacter aerogenes which were unable to synthesize and accumulate high molecularweight polyphosphates (Harold and Harold, 1963). Mutants of the cyanobacterium Anacystis nidulans, deficient in polyphosphates, were recently obtained by Vaillancourt et af. (1978). These mutants could grow, though very poorly, under certain cultivation conditions.

C . METHODS OF DETECTION, IDENTIFICATION A N D FRACTIONATION OF INORGANIC POLYPHOSPHATES

The oldest and most extensively used methods for determining condensed phosphates in biological materials, although of course the least accurate, are based on staining of cells and tissues by certain basic dyes, such as toluidine blue, neutral red and methylene blue. The presence of condensed phosphates in the organisms is judged from the appearance in cells of metachromatically stained granules, or, as they are also known, volutin granules (Kuhl, 1974; Kulaev, 1979). It must be said, however, that, despite the fact that in most cases the cytochemicaldetection of polyphosphate granules is associated with the actual presence of condensed phosphates in the organism, the use of such methods must nevertheless be attended with great caution (Martinez, 1963). This is primarily due to the fact that the basic dyes used to identify polyphosphate granules are also capable of metachromatically staining other polymeric compounds which are encountered in biological material. However, in recent years, primarily owing to the works of Jensen and his coworkers, cytological methods of detecting polyphosphate granules in situ have been significantly improved (Jensen, 1968,1969;Jensen and Sicko, 1974; Sicko-Goad et al., 1975, 1978; Lawry and Jensen, 1979; Baxter and Jensen, 1980a,b). In these works, very interesting results on the structure and formation of polyphosphate granules in cyanobacteria were obtained by electron microscopic and cytochemical methods. Particularly impressive data were furnished by electron microscopy combined with X-ray dispersion analysis (Coleman et al., 1972).This method gives the opportunity of reliably detecting phosphate in the electron-dense inclusions detected by electron microscopy, and also makes it possible to establish the nature of cations present in polyphosphate granules and to establish whether they contain organic components. Besides the above works of Jensen and his coworkers, in

POLYPHOSPHATE METABOLISM I N MICRO-ORGANISMS

a7

a number of studies this method was successful for detection and chemical analysis of polyphosphate granules in various organisms (Jones and Chambers, 1975;Wool and Held, 1976; Kessel, 1977; Hutchinson et al., 1977; Peverly et al., 1978; Adamec et al., 1979; Barlow et al., 1979; Tillberg et al., 1979; Doonan et al., 1979). All these works revealed that the composition of polyphosphate granules changed markedly depending on the chemical and, in the first place, ionic composition of the cultivation medium. However, strictly speaking, this method of polyphosphate detection is not universally appropriate. Firstly, in granules it identifies the mere presence of phosphate but not phosphoryl groups linked by anhydride bonds. Secondly, it does not detect polyphosphates in cells if their concentrations in the subcellular structures is not high enough. A more sensitive and convenient method of polyphosphate detection in situ is by fluorescencemicroscopy using fluorochrornesof the type 4‘,6‘-diamidin0-2-phenylindole-2HCl(DAPI; Allan and Miller, 1980). To date, high-resolution 31P nuclear magnetic resonance has proved to be efficient in detecting intracellular polyphosphates containing phosphate residues linked by anhydride bonds, or so-called “middle” phosphate groups (Glonek et al., 1971; Salhany et al., 1975; Burt et al., 1977; Navon et al., 1977a,b; Ugurbil et al., 1978; Ferguson et al., 1979; Sibeldina et al., 1980; Ostrovsky et al., 1980). Thus, at present, various physical methods are efficiently used for detection of polyphosphates in situ. Moreover, as stated above (Kulaev, 1979), a number of chemical methods are currently employed to identify exactly polyphosphates in extracts from biological material. Of these, the most widely used are chromatographic methods, particularly thin-layer chromatography (Kulaev and Rozhanets, 1973;Kulaev et al., 1974a;Ludwig et al., 1977;Lusby and McLaughlin, 1980; Guerrini et at., 1980; Solimene et af., 1980). However, these methods are applicable only for identification and analytical separation of low molecular-weight polyphosphates between two and seven residues, whereas higher molecular-weight polyphosphates are practically unresolvable. Our preliminary data (I. S. Kulaev, K. G. Skryabin, P. M. Rubtsov and V. D. Butukhanov, unpublished results) suggest that the chromatographic techniques widely used at present for fractionation of oligonucleotides (Maxam and Gilbert, 1977) may prove expedient for separating highly polymerized polyphosphates. As this method combines chromatography and radioautography of 32P-labelled products, it may be considered as a rather promising and sensitive method not only for detecting polyphosphates in biological material, but also for determinating their chain length. The most accurate, though a rather painstaking, method of identification of condensed polyphosphates by the specific product of their partial hydrolysis, cyclic trimetaphosphate, still remains important (Thilo and Wieker, 1957;

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

Kulaev, 1979). It is regretable that only a few researchers resort to this method for strict identification of condensed polyphosphates in extracts from biological material. Enzymic methods for rigorous identification of inorganic polyphosphates in biological objects would surely prove to be less time-consuming and more reliable. However, up to the present, researchers do not yet have at their hands reliable preparative techniques for obtaining pure and stable enzymes of polyphosphate metabolism which could be used as specific reactants for the detection of inorganic polyphosphates. Special mention should be made of several specific methods of analytical determination of inorganic pyrophosphate reported in recent years (see e.g. Putnins and Yamada, 1975). As will be indicated later, use of these methods disclosed certain specific metabolic features of this simplest representative of inorganic polyphosphates in micro-organisms(Mansurova et af., 1975a, 1976; Shakhov et af., 1978; Ermakova et al., 1981). As to the methods of extraction of inorganic polyphosphates from biological material and their fractionation, no new techniques have been reported lately. Of all the available methods of polyphosphate fractionation (Kulaev, 1979) the most informative is that of Langen and Liss (1958), which proved to be expedient owing to the fact that it produced fractions characterized by different intracellular localization and physiological activity (Alking et af., 1977).This method consists in successive extraction in the cold with 5% trichloroacetic acid or occasionally with 0.5 M perchloric acid (acid-soluble fraction Polyp,), saturated sodium perchlorite solution (saltsoluble fraction PolyP2), dilute sodium hydroxide solution (pH 10; alkalisoluble fraction PolyP3) and a more concentrated solution of alkali (0.05 M sodium hydroxide; alkali-soluble fraction PolyP~).However, for a number of organisms, these sequential treatments do not ensure complete extraction of polyphosphates from the cells. Kulaev et al. (1966) suggested extracting the remaining polyphosphates with hot perchloric acid, thus hydrolysing them to orthophosphate. The use of less convenient schemes of fractionation for extraction of polyphosphates from biological materials has meagre prospects, since this is quite often connected with difficulties in interpreting data on polyphosphate metabolism. As indicated below, various fractions of polyphosphates have different pathways of synthesis and degradation. Before concluding the introductory part of this review, it should be pointed out that recent work (see e.g. Baltscheffsky and Stedingk, 1966; Mansurova et al., 1973a,b; Reeves, 1976; Wood, 1977) has unambiguoudy pointed to an essential difference in metabolic pathways and physiological activity between condensed polyphosphates and inorganic pyrophosphate. In this connection, it seems appropriate to discuss some peculiarities of their metabolism.

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

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11. High Molecular-Weight Polyphosphates A. INTRACELLULAR LOCALIZATION

Not long ago, it was postulated that high molecular-weight polyphosphates are localized in the microbial cell within the so-called metachromatic granules or volutin granules (Wiame, 1949; Ebel, 1951; Kuhl, 1960; Kulaev and Belozersky, 1962). This concept applied to both prokaryotes and eukaryotes. A wealth of data reported in recent years shed new light on the intracellular localization of these compounds. The most profound studies in this field have been conducted in eukaryotes. Therefore, we shall begin the discussion with these organisms. 1. Eukaryotes Since the intracellular localization of polyphosphates in eukaryotes is best studied for yeast and fungi, let us in the first place consider data pertaining to these organisms. First indications that, at least in yeast, not all polyphosphates are present inside cells in volutin-like granules were obtained by Weimberg and Orton (1965), Weimberg (1970) and Souzu (1967a,b). Their data suggested that a portion of high molecular-weight polyphosphates were localized on the cell surface, in the region of cytoplasmic membrane. Further progress in this field was related to research conducted in a number of laboratories on polyphosphate metabolism in the fungi Neurospora crassa (Kulaev et al., 1966, 1970a,b; Krasheninnikov et al., 1967, 1968;Trilisenko el al., 1980) and Endomyces magnusii (Kulaev et al., 1967a,b; Afanas’eva et al., 1968; Skryabin et al., 1973; Ostrovsky et al., 1980), as well as in yeast (Indge, 1968; Vagabov et al., 1973; Urech et al., 1978; Diirr et al., 1979; Wiemken et al., 1979; Martinoia et al., 1979; Cramer et al., 1980; Tijssen et al., 1980; Lichko et al., 1982). Following Weimberg and Orton (1965), we studied localization of various polyphosphate fractions by a modified method of Langen and Lis (1958). For this purpose, protoplasts (sphaeroplasts) were isolated from mycelia of N. crassa and cells of E. magnusii, and pure and fairly intact nuclei and mitochondria were obtained from these organisms (Kulaev et al., 1970c,d;Skryabin et al., 1973). Table 1 summarizesthe data obtained by analysing the localization of various fractions of polyphosphates in cells of E. magnusii normally grown for 12 hours and in the same kind of cells cultivated for four hours under conditions of “polyphosphate overplus” after six hours of phosphorus starvation. It can be seen that, after removal of the cell wall, the amount of polyphosphates decreased by 2530% in both types of cells. The

IGOR S. KULAEV AND VLADlMlR M. VAGABOV

90

most condensed polyphosphates extractable by hot perchloric acid (PolyPs) disappeared in both cases. Concentrations of the alkali-soluble (Polyp3+ PolyP4) and salt-soluble (PolyPz) fractions were found to decrease significantly, whereas concentration of acid-soluble polyphosphates (PolyP~) slightly increased. The data obtained showed that an important portion of polyphosphates is localized on the surface of the cell either outside the plasma membrane or closely adjacent to it. Polyphosphates lost in the course of cell-wall lysis are hydrolysed therewith to orthophosphate. This suggests that they are apparently in close contact with polyphosphatases which hydrolyse them during protoplast preparation. The results in Table 1 clearly demonstrate that various fractions of polyphosphates differentially extracted from E. magnusii are localized in different cell compartments. Thus, these results show that different extractability of specific polyphosphate fractions is conditioned by different topographic and chemical compartmentation of polyphosphates in the cell. It follows from the data in Table 1 that high molecular-weight polyphosphates are absent from mitochondria of E. magnusii, and the only fraction detected in nuclei is the salt-soluble one. Similar data were obtained in our laboratory using the same methodological approach for mycelia of N. crassa (Kulaev et af., 1966, 1970d; Krasheninnikov et af., 1967, 1968). The presence of 30-35% polyphosphates in the peripheral parts of yeast cells was repeatedly shown in our laboratory for Saccharomyces carfsbergensis. As seen from Table 2, the most highly polymerized alkali-soluble TABLE 1. Contents of various polyphosphate fractions in Endomyces magnusii (whole cells, protoplasts, mitochondria, and nuclei; mg phosphorus (g dry cells-')). A are data for cells grown in phosphate-sufficient medium, B for cells grown in phosphate-rich medium. After Kulaev et al. (1967a), Afanas'eva et a f . (1968) and Skryabin er al. (1973) Conditions of culture growth B A Phosphorus-containing Whole MitoWhole compounds cells Protoplasts chondria Nuclei cells Protoplasts Acid-soluble PolyPl Salt-soluble PolyPl Alkali-soluble Polyp3 Polyp4 Hot perchloric acid extract PolyPs High molecular-weight polyphosphates (total)

+

0.2 0.7 0.9

0.7 0.4 0.4

0.0 0.0 0.0

0.4 0.0

11.2 3.4 3.3

12.1 1.4 0.8

0.2

0.0

0.0

0.0

2.5

0.0

2.0

1.5

0.0

0.4

20.6

14.3

-

PO LY PHOSPHATE M ETABOLlSM IN MICRO-0 RGANI SMS

91

TABLE 2. Contents of polyphosphate fractions in whole cells and protoplasts of Saccharomyces carlsbergensis. After Vagabov et al. (1973)

Phosphorus-containing compounds Acid-soluble (PolyP1) Salt-soluble (PolyP1) Alkali-soluble (pH 8-10; Polyp,) Alkali-soluble (pH 12; Polyp4) High molecular-weight polyphosphates (total)

Content (pg phosphorus (g wet cells)-’) Whole cells

Protoplasts

706 516 208 356

728 299 23 0

1786

1050

polyphosphate fractions of this yeast are also removed on lysis of cell walls by the “snail” enzyme and are absent from resulting protoplasts (Vagabov et al., 1973). The fact that a substantial portion of highly polymerized polyphosphates is localized in yeast and fungi on the cell surface was directly or indirectly shown in works of other researchers. Van Steveninck and his associates (Jaspers and van Steveninck, 1975; Tijssen et al., 1980) provided cytochemical and biochemical evidence for the presence of highly polymerized polyphosphates on the surface of the yeast cell, outside the plasma membrane. It was also found that, in the logarithmic growth stage, this highly polymerized surface fraction of polyphosphates accounted for up to 40% of the total amount of these compounds in yeast, whereas in the stationary phase, it accounted for only 9%. The rates of turnover of two pools of inorganic polyphosphates detected by these authors (the intracellular pool and the one residing outside the cytoplasmic membrane) differed dramatically. The difference in the chain length and turnover rates of the yeast polyphosphates in these two fractions also supports, albeit indirectly, the concept of their dissimilar compartmentation. Recent data of Trilisenko et af. (1980) speak in favour of a common localization of high molecular-weight polyphosphates and polyphosphatase on the surface of cells of N. crassa. In this work, investigation of polyphosphate turnover in a “leaky” mutant of N. crussa showed that a drastic decrease in polyphosphatase activity in the mutant leads to a substantial accumulation of highly polymerized polyphosphates. This can be clearly seen from the data in Table 3. The results of studies on phosphorus metabolism in a slime mutant of N. crussa (Trilisenko et al., 1982), as well as in the plasmodia of Physurzun polycephalum (Sokolovsky and Kritsky, 1980), suggest the localization of the most polymerized fractions of polyphosphates outside the fungal plasma membrane. In both cases the most polymerized fractions (PolyP3, Polyp4and

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

TABLE 3. Contents of polyphosphatefractions in Neurospora crassa during maximum polyphosphatase activity. Data are shown for strain ad-6 and its leaky mutant 30, 19-3. After Trilisenko et al. (1980) Contents (jig (g dry wt-I)) Phosphorus-containing compounds

A ad-6

B 30, 19-3

B/A (%)

Acid-soluble (PolyP~) Salt-soluble (PolyPz) Alkali-soluble (Polyp3 +PolyP4) Hot perchloric acid extract (PolyPs) Orthophosphate Total (Polyp3 PolyP4+ Polyps) High molecular-weight polyphosphates (total)

2,370 1,920 1,700 200 700 1,900

1,550 2,080 3,000 1,200 720 4,200

65 108 176 600 102 220

6,190

7,830

126

+

Polyps) were absent from fungal protoplasts devoid of cell walls. These data prove the presence of these polyphosphate fractions in the periphery of the cell walls of fungi and yeast. As far as intracellular polyphosphates are concerned, they appear to occupy several cell compartments in these organisms. As already mentioned (see Table l), a portion of the salt-soluble polyphosphates PolyPz was detected in nuclear preparations of E. magnusii (Skryabin et al., 1973) and N . crassa (Kulaev et al., 1970a). The presence of specific fractions of high molecularweight polyphosphates in nuclei of different origin has been demonstrated by many researchers (Penniall and Griffin, 1964; Goodman et al., 1968, 1969; Sauer et al., 1969; Bashirelashi and Dallam, 1970; Mansurova et al., 1975b; Hildebrandt and Sauer, 1977; Sokolovsky and Kritsky, 1980; Offenbacher and Kline, 1980). Sokolovsky and Kritsky (1980) reported interesting findings confirming the occurrence in nuclei of Physarum polycephalum of salt-soluble polyphosphates and also of a certain portion of acid-solublepolyphosphates. Attempts were made to detect the localization of polyphosphates inside nuclei. As found in our laboratory (Mansurova et al., 1975b) in rat liver nuclei, the high molecular-weight polyphosphates exhibiting positive metachromatic reaction occur in fractions of nuclear globulin, histones and acid proteins (Zbarsky, 1970). On the other hand, Offenbacher and Kline (1980) showed that in the same organelle polyphosphates were linked to non-histone proteins. Hildebrandt and Sauer (1977) pointed to the occurrence of high molecular-weight polyphosphates in nucleoli of Physarum polycephalum, i.e. in the site of synthesis of RNA and ribosomes. As already indicated, thoroughly purified and fairly intact mitochondria from E. magnusii (Afana-

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

93

s'eva et al., 1968; Kulaev et al., 1970a,c)and N.crassa (Krasheninnikov et al., 1968; Kulaev et al., 1967b, 1970a,c),unlike nuclei, lack high molecular-weight polyphosphates. Italian researchers (Solimene et al., 1980) recently reported that, in yeast possessing respiring mitochondria at the stage of exponential growth, a distinct peak was detected indicative of the accumulation of low molecularweight polyphosphates with a length of three to eight residues. No such peak could be revealed at this stage of growth in non-respiring yeast. However, these results may be interpreted to indicate that such polyphosphates form and accumulate in respiring yeast not in the mitochondria proper, but rather outside the organelle, from ATP and pyrophosphate (Mansurova et al., 1975a).It is noteworthy that high molecular-weight polyphosphate are absent not only from mitochondria but also from other structures related to energy generation in eukaryotic chloroplasts. This was shown for chloroplasts of Acetabularia mediterranea (Rubtsov et al., 1977) and higher plants (Valikhanov and Sagdullaev, 1979).On purification of A. mediferraneachloroplasts in a sucrose density gradient, the peak of metachromatically stained labile phosphorus compounds was quite distant from the chloroplast fraction (Fig. 2). Electron microscopy revealed that chloroplasts are well preserved under

i3

1000

< -

5.

u

c

.-c 500 'z u 0 .-0

0 0

0

0 Number of fractions

FIG. 2. Distribution of chlorophyll ( 0 pg fraction-'), radioactivity (A pulse min-' fraction-'), and compounds which give metachromatic staining (0)during centrifugation of Acetabularia mediterranea chloroplasts in a gradient of sucrose concentration (0.5-1.5 M, 50,00Og,60 minutes). After phosphorus starvation cells were transferred to a medium containing radioactive phosphate before chloroplasts were isolated. From Rubtsov et al. (1977).

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IGOR S. KULAEV AND VLADIMIR M. VAGABOV

these conditions. These results testify that metabolism and physiological role of high molecular-weight polyphosphates are not directly connected with the respiratory and photosynthetic phosphorylation which are known to operate in mitochondria and chloroplasts. In previous reviews (Kulaev, 1975, 1979) the problem of the occurrence of specific polyphosphate fractions in vacuoles and vesicles of the endoplasmic reticulum was not discussed in detail. However, this question is essential and is at present widely debated in the literature. Its importance stems from the fact that vacuoles and vesicles of the endoplasmic reticulum are specific compartments of eukaryotic cells. Therefore, it is of special interest to provide evidence for the occurrence of high molecular-weight polyphosphates in these subcellular structures of eukaryotes. Indge (1968) was the first to indicate the presence of high molecular-weight polyphosphates in yeast vacuoles. The next step in the investigation of polyphosphate metabolism in yeast vacuoles was initiated by the work of Matile and his associates (Matile, 1978; Urech et al., 1978; Durr et af., 1979; Wiemken el al., 1979; Martinoia et al., 1979; Huber-Walchli and Wiemken, 1979). Employment of methods developed by Wiemken and his colleagues for obtaining purified preparations of intact vacuoles and differential extraction of the cytoplasmic and vacuolar pools of ions and compounds from yeast protoplasts has confirmed the occurrence of inorganic polyphosphates in these cellular structures (Urech er al., 1978; Durr et af., 1979). Of great importance is the conclusion drawn by these authors that, in the course of fractionation, nearly all polyphosphates contained in yeast protoplasts are found in the “gross particulate fraction” which includes mainly vacuoles, nuclei and mitochondria. Though protoplasts contained only about 80% of cellular polyphosphates, the authors inferred that all or nearly all of these compounds were contained in the vacuoles of yeast cells. This conclusion seems to be somewhat erroneous, since polyphosphates disappearing from yeast cells during preparation of protoplasts could be readily degraded to orthophosphate by the closely localized polyphosphatase (see Section II.B, p. 110). Besides, it should be taken into account that nuclei which could also contain some polyphosphates were present in the “gross particulate fraction” and probably to some extent in the purified vacuolar fraction. However, with these reservations in mind, it should be accepted that in the yeast studied by Wiemken et al. (1979) most polyphosphates were present in vacuoles (and possibly in vesicles of the endoplasmic reticulum). Important data were obtained by Diirr et al. (1979) on the chain length of polyphosphates present in yeast vacuoles. As can be seen from Fig. 3, the polyphosphates contained in the vacuoles of yeast cells fall into two fractions on the basis of their chain lengths. The first fraction comprises polyphosphates with a chain length (if) of five units, and the second one had ii values of

PO LY PH0sPHATE M ETA6 0 LI S M IN MIC R 0- 0 R GA NI S MS

-

2.0

95

I-

In

c c W

0 5 .-

a U m W

c

c 0 n c 0

n

v/v,

FIG. 3. Sephadex G-75 filtration of vacuolar polyphosphate. The insert shows a calibration of the column with synthetic polyphosphate of known chain length. From Durr et al. (1979).

15 to 25. This fact, together with the well-known data of Langen and Liss (1958), suggest that in the work of Wiemken e f al. (1979) one part of the vacuolar polyphosphates of yeast may belong to the acid-soluble polyphosphate (Z=5) class and the second to the salt-soluble class (Z= 15-25). These data are in a good agreement with the results of Solimene et al. (1980) who detected substantial amounts of low molecular-weight polyphosphates (Z= 3-8) in stationary respiring and non-respiring yeast, tripolyphosphates being the most abundant. Lusby and McLaughlin (1980) recently detected large quantities of tripolyphosphate (1.8 pmol (lo4cells)-') in Saccharomyces cerevisiae. The concentration of polyphosphates decreased with increasing chain length. However, such a situation is far from being universal in yeasts. Quite recently, French researchers (Beckerich et al., 198I ) failed to detect appreci-

96

IGOR S. KULAEV AND VLADlMlR

M. VAGABOV

able quantities of low molecular-weight polyphosphates in Saccharomycopsis lipolytica. About 40% of the total polyphosphates accounted for had chain lengths of three or five units, and the remainder was salt-soluble. It is not excluded that {hese salt-soluble polyphosphates of S. IipoIytica were also localized preponderantly in vacuoles. The presence of polyphosphates in yeast vacuoles was also estabished with the help of a specific fluorochrome (Allan and Miller, 1980). We also attempted to detect polyphosphates in the vacuole pool of Saccharomyces carlsbergensis using Wiemken's method of differential extraction (Lichko et al., 1982). Our data, summarized in Table 4, suggest that the major portion of the polyphosphates is found in the vacuolar pool. It is noteworthy that polyphosphates are most abundant in vacuoles under conditions of polyphosphate overplus, preceded by a period of phosphorus starvation. Nonetheless, in all cases, some polyphosphates were also detected in other cellular compartments. However, in all studies carried out by the differential extraction method, it remained obscure which cellular compartments contributed to the so-called vacuolar pool. In other words, does the latter term imply only the vacuoles as such, meaning by this the lytic compartment of yeast or other eukaryotic cells as postulated by Matile (1975, 1978), or does it also include vesicles of the endoplasmic reticulum which perform a quite different function in cells of

TABLE 4. Influence of the composition of cultivation medium on the content of phosphorus compounds in Saccharomyces carlsbergensis. After Lichko et al. (1 982) Content (pmol (g wet cells-')) Yeast transferred from complete medium to fresh complete medium (5 hours' growth) Cytoplasm orthophosphate polyphosphate Vacuoles orthophosphate polyphosphate Total cell phosphate

Yeast transferred from phosphate-deficient to fresh complete medium (5 hours' growth)

Yeast transferred from phosphatedeficient medium to fresh phosphate deficient medium (5 hours' growth)

0.65 1.94

0.97 2.90

0.01 1.29

13.71 23.55 191.29

16.29 88.87 321.61

17.10 17.42 141.94

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yeast and other organisms? These vesicles were shown not only to contain polyphosphates (Shabalin et al., 1978,1979), but also to have a specificsystem for their biosynthesis related to formation of glycoproteins (see p. 123). Besides yeast, a substantial portion of polyphosphates was recently shown to occur in the form of intravacuolar polyphosphate granules in other eukaryotes. Cramer et al. (1980) found that, in the N. crassa strain studied, a prominent portion of polyphosphates (at least 50%) was contained in vesicles and vacuoles. The presence of polyphosphate granules was established by electron microscopic examination (see Fig. 4) in cells of the slime mould Dictyostelium discoideum (Gezelius, 1974) and in zoospores of a parasitic aquatic fungus Rosella allomycis during cyst formation (Wool and Held, 1976). Soon after

FIG. 4. Spore of Dictyostelium discoideum with numerous polyphosphate deposits, mainly in small vacuoles. Granules are also seen in elongated vacuoles (upper left) and in two crenate mitochondria (arrows). The section was held in the electron beam to evaporate some of the polyphosphate in the granules. The section was treated with glutaraldehyde, osmium tetroxide and uranyl acetate. Magnification x 66,000.From Gezelius (1974).

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this, the presence of polyphosphate granules in vacuoles of acquatic fungi was confirmed in Blastacladiella emersonii by Hutchinson et al. (1977). In this work, use was made of the X-ray dispersion micro-analytical detection of phosphorus in combination with electron microscopy. This method was first used by Coleman et al. (1972) for detecting polyphosphates in granules in Tetrahymenapyriformis. Using the same technique, polyphosphate granules were detected in vacuoles of Chlorellasp. (Atkinson et al., 1974;Peverly et al., 1978; Adamec et al., 1979)and in Scenedesmus sp. (Tillberg et al., 1979,1980). In the latter organism intravacuolar polyphosphate granules had been detected earlier by cytochemical methods (Sundberg and NilshammarHolmvall, 1975). It can, therefore, be stated that, in eukaryotic cells, a substantial portion of cellular polyphosphates is deposited in the form of polyphosphate granules. However, it remains obscure whether the cell sap of these organisms contains soluble polyphosphates which are isolated from the environment only by the plasma membrane. It appears probable that a portion of the least polymerized polyphosphates happens to be in a free state in the yeast protoplasm. However, such a suggestion should be given experimental support. Results of Ostrovsky et al. (1980), obtained by a 31Pnuclear magnetic resonance 145.78 MHz method of high resolution, point to the possible existence of such a mobile free pool of polyphosphates in Endomyces magnusii (see Fig. 5 ) . It should be noted that the 31Pnuclear magnetic resonance method of high resolution, which is widely employed nowadays for detecting various

I

-30

I

-20

I

-10

I

0

I

10

I

20

I

30

Chemical shift (p.p.m. relative to trimethylphenyl phosphoiadyl)

FIG. 5. A 145.87 MH~-~*P-nuclear magnetic resonance spectrum of cells of Endomyces mangusii (A) with integral intensity (B). Assignment of signals: 1, standard; 2, sugar phosphates; 3,4, intra- and extracellular orthophosphate of hydrocarbons; 5, 6, y-phosphate of nucleoside tri- and diphosphates; 7, a-phosphates of di- and triphosphates; 8, dinucleotides NAD+ and other compounds; 9, derivatives of nucleoside diphosphates; 10, #I-phosphate of nucleoside triphosphate; 11, pofyphosphate. From Ostrovsky et al. (1980).

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phosphorus-containing compounds in cells (Solhany et al., 1975; Burt et al., 1977; Navon et al., 1977a,b), has some limitations. It allows various phosphorus-containing compounds, such as ATP, sugar phosphates and polyphosphates, to be detected only when they occur in cells in a free state. If they are linked, as for example in polyphosphates, to other cellular components, such as proteins or nucleic acids, then they may not be detected by this method. Therefore, conventional chemical analysis should be conducted in parallel with investigations aimed at the quantitative assessment of intracellular polyphosphates by the 31Pnuclear magnetic resonance method of high resolution. Unfortunately no such results are available in the literature to date. 2. Prokaryotes Prokaryotic cells have much simpler structures compared with the simplest eukaryotes, such as yeast, fungi or algae. First of'all they have no nucleus enveloped by a membrane. Instead they have a nucleotid containing DNA strands and nucleoplasm. Moreover, prokaryotes, in particular eubacteria and cyanobacteria, do not contain vacuoles. As for autotrophic bacteria and cyanobacteria, they possess thylacoids (specialized protrusions of the plasma membrane, in which the photosynthetic apparatus of these organisms is localized) and the so-called polyhedral bodies, or carboxysomes, which harbour, according to Stewart and Codd (1975), the key enzyme of photosynthesis ribulose 1,5-diphosphate carboxylase. When discussing localization of high molecular-weight polyphosphates in prokaryotic cells, it should be noted that the latter do not possess the two compartments that eukaryotes have for storing the bulk of intracellular polyphosphates, namely the nucleus proper, limited by the nuclear membrane, and vacuoles. Then where are polyphosphates accumulated in prokaryotic cells? Polyphosphate-containinggranules, or volutin granules, have been unequivocally demonstrated in bacteria, in particular in Spirillum uolutans (Ebel et al., 1958; Drews, 1962; Hughes and Muhammed, 1962; Kulaev and Belozersky, 1962). Various cytochemical methods were elaborated for detecting volutin-like granules in different micro-organisms(Keck and Stich, 1957;Ebel et al., 1958; Talpasayi, 1963). Cytological methods for detecting polyphosphate granules were boosted by the use of the electron microscope (Niklowitz and Drews, 1957; Ebel et al., 1958; Ris and Singh, 1961; Drews, 1962; Jost, 1965; Voelz et al., 1966; Friedberg and Avigad, 1968;Jensen, 1968, 1969). For early references on detection of polyphosphate granules we recommend the reader to refer to Kuhl (1960, 1962, 1974), Drews (1962), Harold (1966), Shively (1974) and Kulaev (1979).

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In recent years, the most important and comprehensive data in this field were obtained by Jensen and his coworkers (Jensen, 1968, 1969; Jensen and Sicko, 1974; Sicko-Goad et al., 1975; Sicko-Goad and Jensen, 1976; Lawry and Jensen, 1979; Baxter and Jensen, 1980a,b). These works were carried out with cyanobacteria which at present are given much attention because of the necessity of solving a number of applied problems concerning water pollution. Special attention is paid to the possible use of blue-green algae as accumulators of substantial amounts of phosphates in the form of polyphosphates. This problem arose from severe pollution of inland water bodies, especially in industrialized countries, with various detergents of which sodium tripolyphosphate is the most abundant pollutant. Using electron microscopy with the cyanobacteria Nostoc prunifarme (Jensen, 1968), Plectonema boryanum (Jensen, 1969; Jensen and Sicko, 1974; Sicko-Goad and Jensen, 1976) and Anacystis nidulans (Lawry and Jensen, 1979), Jensen and his colleagues investigated the accumulation of polyphosphate granules under various cultivation conditions. In these experiments special emphasis was laid on the accumulation by cyanobacteria of polyphosphate granules under conditions approximating those of inland water bodies, i.e. conditions of phosphorus and sulphur starvation. Waters in such reservoirs are known to contain about 0.01 pg phosphorus I - ’ (Jensen and Sicko, 1974), and this creates conditions of phosphorus starvation for micro-organisms. When large amounts of industrial and domestic detergents, in particular tripolyphosphate, enter inland water bodies, an intensive “fluorescence” of cyanobacteria occurs leading to contamination of vast reservoirs of drinking water. This has become quite a serious problem, and many laboratories all over the world are endeavouring to solve the problem. From electron microscope studies on the localization of polyphosphate granules in Plectonema boryanum cultured in medium containing the normal content of phosphorus, as well as under conditions of phosphorus starvation followed by subsequent “phosphate overplus” in medium enriched with phosphorus, Jensen drew the following conclusions (Jensen and Sicko, 1974). In normal growth conditions, polyphosphate granules are found mainly on DNA fibrils and in a zone enriched with ribosomes. Under conditions of phosphorus starvation, in addition to these sites there was a zone of average electron density formed in the region of nucleoplasm, apparently as the result of degradation of a portion of nucleic acids. Under conditions of “phosphate overplus”, polyphosphates accumulated in the region of nucleoplasm, and polyphosphate granules appeared in the polyhedral bodies directly involved in the dark reactions of photosynthesis in cyanobacteria (Stewart and Codd, 1975). In certain cells, polyphosphate granules formed near thylakoids which in these organisms contain chlorophyll and perform phosphorylation reactions.

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Thus electron microscopy helped to establish that, in blue-green algae, polyphosphate granules are localized in most cases in the region of nucleoid (DNA fibrils and nucleoplasm) rich in ribosomes and near the subcellular structures participating in photosynthesis. Similar reports for cyanobacteria were made by other authors using the same approach (Vaillancourt et al., 1978; Barlow et al., 1979).The data obtained, at least those for localization of polyphosphate granules in the vicinity of the bacterial nucleoid, correlate well with previous findings using the same method on heterotrophic prokaryotes (Drews, 1962;Voelz et al., 1966; Friedberg and Avigad, 1968; Deinema et al., 1980). However, the data of Jensen and other cytologists have one shortcoming; there was no chemical determination of polyphosphates carried out in parallel with the electron microscope studies. In some studies, Jensen (Sicko-Goad and Jensen, 1976) attempted to compare the electron microscope picture with accumulation of total phosphorus in certain fractions. Still, this is evidently insufficient for allowing a rigorous conclusion to be made about the polyphosphate nature of granules detected in cells. As was referred to in the Introduction, Kessel (1977) and Jensen and his coworkers (Sicko-Goad et al., 1975; Baxter and Jensen, 1980a) combined electron microscopy with X-ray energyaispersion microanalysis(Colemanet al., 1972)to reveal the chemical nature of these granules. This method enables one to locate in situ phosphorus, sulphur, calcium, potassium and carbon dioxide in specific cellular compartments. However, this method does not provide information as to the forms of compounds of phosphorus, carbon and other elements occurring in the cellular inclusions. Nevertheless, the results obtained with this technique allow one to establish the phosphate nature of granules and to determine which cations may be present in these granules. In his recent investigations, Jensen (Baxter and Jensen, 1980a,b) showed that, under ordinary cultivation conditions, appreciable amounts of potassium and comparatively low quantities of calcium and magnesium are present, in addition to phosphorus, in polyphosphate granules of the cyanobacterium Plectonema boryanum. Under special conditions, when the medium contains an excess amount of a particular metal, such as Mg2+,Ba2+,Mn2+or Zn2+, they accumulate in large quantities in polyphosphate granules. Strontium is also known to be able to accumulate in considerable amounts in cells of these algae, not in polyphosphate granules, but in some inclusions containing, together with K + and Ca2+,sulphur instead of phosphorus. Useful information on localization of polyphosphates in bacterial cells may be provided by 31Pnuclear magnetic resonance of high resolution at 145.78 MHz (Ferguson et al., 1979; Ostrovsky et al., 1980). Ostrovsky et al. (1980), for example, believed that a marked increase in the intensity of a low-field 3'P nuclear magnetic resonance signal shift for polyphosphates, observed when cells of Mycobacterium smegmatis were treated with ethylene diamine

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tretra-acetic acid, points to localization of certain amount of mobile inorganic polyphosphates in the periplasmic region of Mycobacterium smegmatis, i.e. outside the cytoplasmicmembrane. These findings, though not yet confirmed by other investigators, appear to be extremely important, since in eukaryotes also a portion of polyphosphate is localized outside the cytoplasmic membrane, and only part of polyphosphates occur in the form of polyphosphate granules inside the cell. Some recent research on the chemical fractionation of polyphosphates in a number of bacteria also support, albeit indirectly, such a conclusion (Bobyk et al., 1980;Egorova et al., 1981; Nikitin et a)., 1979, 1983). Bobyk and his coworkers isolated and qualitatively assessed various fractions of polyphosphates from Bdellovibrio bacteriovorus, and showed that, in this bacterial parasite, most polyphosphates occur in the form of acid-insoluble highly polymerized fractions. A similar situation was revealed in prosthetic oligotrophic bacteria, namely Tuberoidobacter and Renobacter spp., studied by Nikitin et al. (1979). In contrast, in the thermophilic Thermusflavus growing at 6570°C most polyphosphates were detected in the form of low-polymeric fractions PolyP~and PolyP~.Comparison of these results with similar data obtained for eukaryotes suggests that Bdellovibrio sp. and prosthetic oligotrophic bacteria populating the atmosphere, under conditions of permanent starvation, contain predominantly outwardly localized highly polymerized polyphosphates, whereas in thermophiles, polyphosphates of relatively low molecular weight are mainly localized within the plasma membrane. Similar examples may be found in Kulaev’s (1979) monograph summarizing all previous publications in the field. This book provides data, though rather scanty to date, on isolation in a pure state of polyphosphate granules from cells of some micro-organisms. Recently, Jones and Chambers (1975) succeeded in isolating these granules from Desulfovibrio gigas (Fig. 6). The granules proved to be soluble only in 1 M HCI, and insoluble in water, 1 M NaOH, ethanol, ether and other organic solvents, and appeared to be tripolyphosphate of magnesium (Mg~(P301o)s). It is interesting that in this work, infrared spectroscopy (Corbridge and Lowe, 1955) was employed to establish the precise nature of these granules. Using this and other methods, it was rigorously proved that the granules are composed of magnesium tripolyphosphate. It should be noted that these granules formed only in sulphate-reducing bacteria Desulfovibrio spp. and after repeated inoculations of the culture. The possibility of accumulation of large amounts of magnesium tripolyphosphate in bacterial cells in the form of volutin granules is a novel and very interesting fact. These results correlate, to some extent, with the investigations of Rosenberg (1 966) and Simkiss (1981). In both of these studies (in the first one with the protist Tetrahymena puriformis, and in the second with the

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FIG. 6. Electron micrograph of isolated granules from Desulfovibrio gigus. From Jones and Chambers (1975).

hepatopancreas of the mollusc Helix aspersa) the granules isolated were composed almost entirely of Ca-Mg pyrophosphate. B . ENZYMES INVOLVED IN BIOSYNTHESIS A N D DEGRADATION OF POLYPHOSPHATES

Investigations on the localization of high molecular-weight polyphosphates in cells of various organisms were carried out simultaneouslywith studies on the enzymes involved in their metabolism. 1. Polyphosphate: ADP Phosphotransferase

The enzyme polyphosphate: ADP phosphotransferase (EC 2.7.4. I) was the first to be identified. This enzyme catalyses the transfer of the high-energy

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IGOR S. KULAEV AND VLADlMlR M. VAGABOV

phosphate residue from ATP to polyphosphates and back from polyphosphates to ADP to form ATP. The history of the discovery of this enzyme and its occurrence in various organisms have been described in Kulaev’s monograph (1979). Polyphosphate: ADP phosphotransferase (polyphosphate kinase) was first isolated from Escherichia coli and purified as described by Kornberg et al. (1956). It catalyses the following reaction: ATP+ (polyphosphate),,

polyphosphate kinase

ADP + (polyphosphate),+1

(1)

It was later isolated from other micro-organisms (Kulaev, 1979). After the discovery of the enzyme, some researchers (Hoffmann-Ostenhof, 1962; Y oshida, 1962) began to consider high molecular-weight polyphosphates as peculiar microbial phosphagens, i.e. as compounds that can be synthesized and utilized in micro-organisms only via the ADPctATP system, similarly to creatine and arginine phosphates in animal tissues. However, further studies of enzymic reactions involving high molecularweight polyphosphates have shown the limited nature of this hypothesis on their physiological role in micro-organisms. Moreover, our findings (Kulaev and Rozhanets, 1973; Kulaev et al., 1974a) indicate that both high molecularweight polyphosphates and polyphosphate kinase are present in animal tissues. They have been found in the rat brain, i.e. in the tissue for which the existence of the classic phosphagenic creatine phosphate-creatine kinase system has long been known. This fact, considered as such, suggests that the physiological role of high molecular-weight polyphosphates cannot be confined to the function of common “phosphagens”, particularly “microbial phosphagens”. This statement is corroborated by the detection of this enzyme in yeast vacuoles (Shabalin et al., 1977). Further, Schwencke (1978) reported the presence in these cellular structures of polyphosphate depolymerase (see below) which apparently also plays some role in metabolism of the vacuolar polyphosphate pool. However, it is not excluded that the function of this enzyme in vacuoles is basically connected with transport of polyphosphates through the tonoplast. In some prokaryotic organisms, polyphosphate kinase may possibly stand in the centre of the entire polyphosphate metabolism, and be the key metabolic enzyme. This idea is supported by the fact that, in mutants defective in polyphosphate kinase, such as Aerobacter aerogenes (Harold and Harold, 1963) and Anacystis nidulans (Vaillancourt et al., 1978), accumulation of high molecular-weight polyphosphates stopped. However, in fungi, for example Neurospora crassu, this enzyme probably does not occur at all (Kulaev et al., 1971). Nevertheless, this organism accumulates and metabolizes these compounds. Although polyphosphate: ADP phosphotransferase has been found in the

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slime mould Dictyostelium discoideum, its activity proved to be low at all stages of the differentiation of this organism (Gezelius, 1974). The enzyme did not materially contribute to the process of ATP formation which testifies convincingly against the phosphagenic function of polyphosphates detected in large amount in the fungus (Gezelius, 1974; Al-Rayess et al., 1979). On the other hand, it has been shown in our recent work (Butukhanov et al., 1979) that, in a Corynebacterium sp., in production of ATP from exogenous adenine, polyphosphate: ADP transferase contributes significantly to synthesis of ATP which is accumulated in large amounts (0.6-1 .O mg ml-I) in the culture medium. During the period of maximum synthesis of ATP from exogenous adenine in autolysed cells of the strain, the activity of polyphosphate: ADP phosphotransferase increased greatly. However, it does not follow from these data that the enzyme and its substrate, high molecularweight polyphosphates, are essential for ATP formation inside cells of a normally growing culture of Corynebacterium sp. 2. Polyphosphate: Adenosine Monophosphate Phosphotransferase Soon after detection of polyphosphate kinase in some micro-organisms, Winder and Denneny (1957) found another enzyme which suggested that metabolism of high molecular-weight polyphosphates in micro-organisms could, in many ways, be connected with that of adenine nucleotides. This enzyme, polyphosphate: AMP phosphotransferase, was isolated and partially purified from mycobacteria in Ebel’s laboratory (Dirheimer and Ebel, 1965). The enzyme is responsible for the reaction:

+

polyphosphate: A M P

+

AMP (polyphosphate), , A ADP (polyphosphate),phosphotransferase

1

(2)

Hitherto this enzyme had been found only in mycobacteria and corynebacteria (Kulaev, 1979). Recently, an unsuccessful attempt was made to detect polyphosphate: AMP phosphotransferase in the slime mould Dictystelium discoideum (Gezelius, 1974).On the other hand, the occurrence of this enzyme in corynebacteria and its involvement in ADP synthesis was shown indirectly by Butukhanov et al. (1979) in a Corynebacterium strain that produces ATP from exogenous adenine.

3. Polyphosphate (Metaphosphate)-Dependent NAD + Kinase Quite recently, another enzyme linking high molecular-weight polyphosphates (metaphosphates) with energy metabolism, i.e. polyphosphate (metaphosphate)-dependent NAD+-kinase was detected in some eubacteria by Murata et al. (1980). In the course of studies on the specificity of this enzyme

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for the phosphoryl-group donor, these authors found it to be specific for metaphosphate manufactured by Katayama (Japan). However, as pointed out by Kulaev (1979), chemists estabished that the so-called high molecularweight “metaphosphate” was no more than a linear high molecular-weight polyphosphate of the Graham salt type. Murata et al. (1980) did not investigate specifically “metaphosphate” from Katayama. In this connection, the Japanese researchers seem to have dealt with a preparation of linear polyphosphates. They found polyphosphate (metaphosphate)-dependent NAD+ kinase in species of Acetobacter, Achromobacter, Brevibacterium, Corynebacterium, and Micrococcus, but failed to detect it in species of Escherichia, Proteus and Aerobacter. This enzyme catalyses the following reaction: NAD

+

+polyphosphate (metaphosphate),eNADP +

polyphosphate (metaphosphate),-

I

(3)

This enzyme differs from the ATP-dependent NAD+ kinase in pH optimum, thermostability and a number of other properties. 4. Polyphosphate: D-Glucose 6-Phosphate Phosphotransferase

The Polish scientist Szymona (Szymona, 1962;Szymona and Ostrowski, 1964) detected another enzyme of polyphosphate metabolism, polyphosphate: D-glucose 6-phosphotransferase (polyphosphate-glucokinase,EC 2.7.1.63) in mycobacteria, and showed that it catalysed the specific transfer of the phosphate group from high molecular-weight polyphosphates to glucose to form D-glucose 6-phosphate:

+

polyphosphate-

D-Glucose (polyphosphate), Y-_-D-glucose 6-phosphate + glucokinase

(polyphosphate),-

1

(4)

The discovery of this enzyme in this and related micro-organisms(Szymona et al., 1967; Uryson and Kulaev, 1968; Szymona et al., 1977; Szymona and Szymona, 1979; Eroshina et al., 1980; Ziizina et al., 1981) has shown that high-energy phosphate residues of polyphosphates can be utilized directly without the participation of the ADP-ATP system. This finding has also demonstrated that, in some cases, polyphosphates can perform the function that is normally carried out by ATP itself. The reaction described by Szymona and his colleagues is identical with the classical hexokinase reaction during which glucose undergoes phosphorylation caused by ATP. Szymona and his coworkers, in their recent work on purification and specific functions of polyphosphate glucokinase in Nocardia

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minima (Szymona and Szymona, 1979), Mycobacterium tuberculosis H37Ra (Szymona et al., 1977; Pastuszak and Szymona, 1980), and a number of other mycobacteria (Szymona and Szymona, 1978),showed that electrophoretically homogeneous preparations of polyphosphate glucokinase exhibited certain activity also with ATP as the phosphate donor. However, it is not yet clear whether they dealt in both cases with a mixture of two enzymes having polyphosphate hexokinase and ATP hexokinase activities, or a single protein possessing two different active centres one of which operates with ATP and the other with high molecular-weight polyphosphates. H. G. Wood (personal communication) reported his success in distinguishing between the two activities in the course of isolating polyphosphate hexokinase from Propionibacterium shermanii. However, his highly active preparation exhibited low ATP-hexokinaseactivity. It is noteworthy that, in the above works, Szymona and his colleagues have established the existence in various bacteria of isoforms of polyphosphate glucokinase having different molecular weights. Nocardia minima, for example, was found to have three isoenzymes with molecular weights of 59,000, 76,000 and 150,000, respectively (Szymona and Szymona, 1979).The prevailing fraction, which accounted for 80% of the total polyphosphate glucokinase activity, showed preference for polyphosphate but one of the minor fractions preferred ATP. Summing up, this very important enzyme of polyphosphate metabolism is now receiving the most detailed investigation and, before long, one should expect a breakthrough in understanding of the mechanism of its operation. It is of interest that, in recent years, researchers undertook a number of investigations aimed at detecting polyphosphate glucokinase activity in various organisms, of which special mention should be made of the progress in detecting this activity in a bacterial parasite Bdellovibrio bacteriovorus (Bobyk et al., 1980), as well as in the recently described oligotrophic bacteria Renobacter vacuolatum (Nikitin et al., 1983). Of importance also is the fact that, in Dictyostelium discoideum and other fungi (Kulaev, 1979),this enzyme was not detected (Gezelius, 1974). In conclusion, it should be recalled that Szymona and his coworkers also revealed the existence of a whole series of adaptive enzymes that are responsible for transfer of phosphate group from high molecular-weight polyphosphates to other sugars and their derivatives, namely fructose, mannose and glucuronic acid (Szymona and Szumilo, 1966; Szymona et al., 1969).

5 . I ,3-Diphosphoglycerate: Polyphosphate Phosphotransferase In addition to the enzymes already referred to, another enzyme catalysing synthesis of high molecular-weight polyphosphates was found (Kulaev et al.,

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1968; Kulaev and Bobyk, 1971; Kulaev, 1979). This enzyme, which was first detected in N. crassa and later in other micro-organisms including fungi and eubacteria (Kulaev et al., 1971), is involved in transfer of a high-energy phosphoryl group from 1,3-diphosphoglyceric acid to polyphosphate. Thus, the enzyme, 1,3-diphosphogIycerate:polyphosphate phosphotransferase (EC 2.7.4.17), participates in a reaction similar to the well known reaction of ATP formation during glycolytic phosphorylation.

+

+

1,3-Diphosphoglycerate (polyphosphate),~3-phosphoglycerate

(polyphosphate),+ I

(5)

In our work, it has been shown that this enzymic system is controlled by adenine nucleotides,principally ATP (Kulaev, 1979).This was first revealed in a mutant of N. crassa deficient in adenine synthesis (Kulaev et al., 1968; Kulaev and Bobyk, 1971). In all cases when this enzymic activity was found in micro-organisms, it proved to be fairly low and obviously did not provide for biosynthesis of all polyphosphate fractions (Kulaev e f al., 1971, 1973a). Apparently, in N. crassa, this enzyme participates primarily in metabolism of low molecularweight polyphosphates contained in polyphosphate granules and it was in these granules that it was detected by Kulaev and Konoshenko (1971a). In conclusion, it may be noted that, of all micro-organisms studied, highest activity of the enzyme has been detected in Bdellovibrio bacteriovorus (Bobyk et al., 1980).If one takes into account that polyphosphate glucokinasehas also been found in this organism, then an inference may be drawn about a close relation between polyphosphate metabolism and glycolysis. 6. Polyphosphate Polyphosphohydrolases In addition to the already mentioned phosphotransferases, two types of phosphohydrolases are involved in metabolism of high molecular-weight polyphosphates (Kulaev, 1979). Phosphohydrolases of one type split high molecular-weight polyphosphates within the chain into smaller fragments. These are the so-called polyphosphate polyphosphohydrolases (polyphosphate depolymerase, EC 3.6.1.10). They catalyse the reaction depicted below: (polyphosphate),+ water

polyphosphate

depolymerase

(polyphosphate),-,

+(polyphosphate),

In cells of eukaryotes, namely yeasts and fungi, several different polyphosphate depolymerases cleaving polymers of different lengths in the middle of the chain (Ingelman and Malmgren, 1947, 1948; Mattenheimer, 1956a,b,c;

POLYPH0S PHATE METAB0LISM IN MIC R 0- 0R GAN ISMS

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Kritsky et al., 1972) have been detected. Kritsky et al. (1972) showed that polyphosphate depolymerases which split specific fractions of polyphosphates exhibit their activities at various stages of growth of N. c r ~ ~ s a . Therefore, their action on specific fractions of polyphosphates is timed to a definite physiological state of this fungus. The problem of the intracellular localization of polyphosphate depolymerases appears to be of special interest. In our studies on localization of polyphosphate depolymerases which split high molecular-weight polyphosphates (n= 180and290)inN.crassa,itwasshownthat theiractivityismainlyexhibited at the cell periphery. In protoplasts, after removal of the wall, polyphosphate depolymerasesretain about 10-1 5% of their activity on the already mentioned substrates. As to specific cellular structures, activity was found in fractions of nuclei, as well as in vesicles of the endoplasmicreticulum and vacuoles (Kulaev et al., 1972a).These results were recently supplementedby those of Schwencke (1 978) who revealed the presence of polyphosphate depolymerase-splitting polyphosphates of lower molecular weight in yeast vacuoles. It may be suggested that polyphosphate depolymerases play an extremelyimportant role in polyphosphate metabolism linking the pools (fractions)of these compounds in cells, particularly in those of lower eukaryotes. According to Kritsky and Chernysheva ( I 973), polyphosphate depolymerases participate in translocation of specific polyphosphate fractions through cell membranes. The authors believe that the energy released during polyphosphate cleavage may be utilized for translocation of fragmented molecules through membranes. From this point of view, it is not surprising that polyphosphate depolymerase activities were revealed in regions of plasma membrane, nuclei and vacuoles, i.e. at the sites of localization of basic specific pools (fractions) of polyphosphates in cells of lower eukaryotes. 7. Polyphosphate-Phosphohydrolases Another group of polyphosphatases split one terminal phosphate residue from each polyphosphate molecule. Most investigatorsshare the opinion that these enzymes, called polyphosphate phosphohydrolases or simply polyphosphatases (EC 3.6.1.1 l), are responsible for the occurrence of the following reaction:

+

(polyphosphate), water-+(polyphosphate),- I +Pi

(7)

Thus, enzymic breakdown of polyphosphates occurs by processes similar to the enzymic degradation of other biopolymers, for example, proteins and polysaccharides. Polyphosphate depolymerase and polyphosphatase are, in other words, endo- and exopolyphosphate phosphohydrolases that catalyse

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cleavage of internal and terminal phosphoanhydride bonds. However, the results of numerous studies reported earlier (Kulaev, 1979), as well as quite recent experimental findings, suggest that both mechanism of action and physiological role of these enzymes may differ markedly. Polyphosphatases capable of hydrolysing high molecular-weight polyphosphates to orthophosphate were found in many organisms (Kulaev, 1979).The number of organisms in which these enzymes were found has increased markedly in recent time and includes, among others, Nocardia erythropolia and Brevibacterium sp. (Eroshina et al., 1980), Corynebacterium sp. (Butukhanov et al., 1979), Bdellovibrio bacteriovorus (Bobyk et al., 1980), Streptomyces levoris (Zuzina et al., 1981), Tuberoidobacter mutans and Renobacter vacuolatum (Nikitin et al., 1983), Dictyostelium discoideum (Gezelius, 1974) and Candida guillermondii (Kulaev et al., 1974b).It is noteworthy that a rather high polyphosphatase activity was revealed in many soil micro-organisms (Aseeva et al., 1981), for example, in bacteria of the genera Bacillus and Micrococcus, fungi of the genera Aspergillus and Penicillium and coryneforms of the genus Arthrobacter. The fungi Aspergillus wentii and Cladosporium herbarum released their polyphosphatase into the cultivation medium; in these organismsone would suggest that outside the cells of these fungi, the enzymes are capable of digesting polyphosphates available in soil into orthophosphate. Remarkably, Aseeva et al. (1981) revealed polyphosphatase activity in sterile soil. These authors showed also that many soil organisms could grow intensively on a medium with polyphosphates as the sole form of phosphate. These substrates are degraded therewith by polyphosphatase to orthophosphate which is assimilated. Other researchers succeeded in finding polyphosphatase activity (towards polyphosphate with n =40) in sterile roots of the cotton plant (Valikhanov and Sagdullaev, 1979; Igamnasarov and Valikhanov, 1980). Owing to this activity, cotton plants could assimilate the phosphorus of polyphosphates available in the isolation medium more efficiently than when the same amount of phosphate was available in the form of orthophosphate. These data point to an important part played by polyphosphates of micro-organisms and plants in the phosphorus cycle in the soil, as well as to promising prospects for the use of polyphosphates as phosphorus fertilizers. It may be of interest that we have not detected any polyphosphatase hydrolysingpolyphosphates of high molecular-weight in the phytopathogenic fungus Phytophthora infestans (Sysuev et al., 1978). This may be a specific feature of polyphosphate metabolism in microbe-parasite associations which proliferate in the cells and tissues of plants rather than the soil. The problem of intracellular localization of polyphosphatases appears to be very important. Previous reports (see Kulaev, 1979) as well as recent investigations (Nesmeyanova et al., 1975a, 1976; Severin et al., 1975;

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Trilisenko et al., 1980,1982)indicated that polyphosphatase hydrolysing high molecular-weight polyphosphates to orthophosphate was essentially localized on the outer side of the plasma membrane of eukaryotes and prokaryotes. It was shown that in both Neurospora crassa (Kulaev, 1979)and Escherichia coli (Nesmeyanova et al., 1975a, 1976; Severin et al., 1975) a significant portion of the polyphosphatase was membrane-bound and therefore could be detached only by treatment with detergents, like Triton X-100.However, the polyphosphatases of N. crassa and E. coli differ markedly. In bacteria, polyphosphatase, or rather its biosynthesis and secretion into the periplasm, is repressed by exogenous phosphate (Harold, 1966; Nesmeyanova et al., 1975a,b, 1976; Maraeva et al., 1979; Kulaev, 1979),whereas in fungi this does not occur (Umnov et al., 1974a; Kulaev, 1979; Trilisenko et al., 1981). Investigations of Nesmeyanova et al. (1976) and those of Maraeva et al. (1979) showed that, during derepression of polyphosphatase synthesis in E. coli under conditions of phosphorus starvation, a substantial portion of this enzyme was transferred from the membrane to the periplasm. It was also found that E. coli polyphosphatase had complex regulatory relations with other phosphohydrolases, and some membrane proteins, of the bacteria. This fact is discussed in greater detail on p. 136. When discussing polyphosphatases hydrolysing high molecular-weight polyphosphates to orthophosphate, mention should be made of the detection of multiple forms of these enzymes in eukaryotic micro-organisms. In particular, this was shown for Endomyces magnusii (Afanas’eva el al., 1976) and Neurospora crassa (Trilisenko et al., 1982). It should be noted too that, even in the “leaky” mutant of N. crassa marked by a five-fold decrease in polyphosphatase activity, the same two isozymes are preserved compared with the initial culture. All of the information presented in this section about polyphosphatases concerns only those that hydrolyse high molecular-weight polyphosphates. Our studies on the polyphosphatase from N. crassa showed that this enzyme hydrolysed, with fairly high and nearly the same rates, polyphosphates of different degrees of polymerization (Kulaev, 1979). However, N. crassa was found to possess a specific enzyme that hydrolysed tripolyphosphate to orthophosphate (Kulaev et al., 1972b; Umnov et al., 1974b; Egorov and Kulaev, 1976). It was called tripolyphosphate hydrolase (EC 3.6.1.25). Tripolyphosphatase activity was found in many organisms (Kulaev, 1979). It has also been detected in some bacteria: Nocardia erythropolus (Eroshina er al., 1980), Tuberoidobacter mutans (Nikitin et al., 1983), Renobacter vacuolatum (Nikitin et al., 1983), Escherichia coli (Nesmeyanova et al., 1975b), Streptomyces levoris (Ziizina et al., 198l), Bdellovibrio bacteriovorus (Bobyk et

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

al., 1980),andinaparasiticfungus Phytophthora infestans(Sysuevetal., 1978). The intracellular localization of tripolyphosphatase was investigated in N. crassa (Kulaev et al., 1972b; Kulaev, 1979). This study provided convincing proof that the enzyme is mainly localized in the mitochondria of N . crassa. It should be underlined that, in this fungus, compartmentation of the enzyme under consideration is quite different from that of the polyphosphatase hydrolysing high molecular-weight polyphosphates. It remains obscure where and in which way tripolyphosphatase is localized in cells of prokaryotic organisms. Igamnasarov and Valikhanov (1980) reported a high extracellular tripolyphosphatase activity in sterile cotton seedlings in a medium deficient in phosphate. It is noteworthy that, in some instances, tripolyphosphatase may be absent from cells of micro-organisms. The data of Jones and Chambers (1975) point indirectly to this fact. Without special precautions and using simple techniques, they succeeded in isolating, from the bacterium DesuIfovibrio gigas, polyphosphate granules composed of pure magnesium tripolyphosphate. If tripolyphosphatase were contained in these bacteria, then under the conditions used for isolating the granules the tripolyphosphate would have undoubtedly been hydrolysed. 8. Variations in the Enzymes of Polyphosphate Metabolism in Micro-Organisms

Investigation of the metabolism of high molecular-weight polyphosphates in various organisms has revealed dramatic variations in the sets of enzymes involved (Kulaev, 1979). Polyphosphate hexokinase has so far been detected only in organisms that fall into the actinomycetes classified according to Krasil'nikov (1949; Szymona et al., 1967; Uryson and Kulaev, 1968, 1970; Kulaev et al., 1971, 1973a, 1976; Uryson et al., 1973, 1974; HoStalek et al., 1976; Eroshina et al., 1980; Murata et al., 1980; Ziizina el al., 1981). In addition to these organisms, polyphosphate hexokinase was recently detected in Bdellovibrio bacteriovorus (Bobyk et al., 1980) and an oligotrophic bacterium Renobacter vacuolatum (Nikitin et al., 1983). However, the systematic position of these exotic bacteria is not yet clear. The group of micro-organisms claimed by Krasil'nikov to be actinomycetes, in particular mycobacteria, corynebacteria and propionic bacteria, contain practically all known enzymes of polyphosphate metabolism (Kulaev, 1979). In contrast, Aerobacter aerogenes according to Harold (1966) and Murata et al. (1980), has only two enzymes of this set; these are polyphosphate kinase, involved in synthesis of polyphosphates, and a polyphosphatase, which hydrolyses them to orthophosphate. At the present time, not all

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113

enzymes of polyphosphate metabolism have been detected in eukaryotes. As already indicated, polyphosphate kinase has not been found yet in N.crassa, and polyphosphatase has not been found in Phytophthora infestans. Nevertheless, in most other eukaryotic micro-organisms all enzymes are detectable. Moreover, owing to the more complex compartmentation of cells, and cellular metabolism as a whole, they apparently have more complex enzymic systems for both synthesis and utilization of polyphosphates. This will be discussed later (p. 114). So far, metabolism of high molecular-weightpolyphosphates, the enzymes involved, and their intracellular localization, have been best studied in heterotrophic eukaryotes. In this connection, Fig. 7 shows a schematic localization of different enzymes of polyphosphate metabolism in an abstract cell of a heterotrophic eukaryote. As for autotrophic eukaryotes, their metabolism, as well as enzymes involved in polyphosphate transformations, have not been sufficiently studied. Polyphosphate kinase, for example, has been found only in three representatives of autotrophic organisms; these are

1

4 5 9

2

3

6

10

7

8

FIG. 7. Localization of polyphosphates and enzymes ofpolyphosphate metabolism in a typical cell of heterotrophic eukaryotic micro-organisms. 1, indicates acid-soluble polyphosphates (Polyp& 2, salt-soluble polyphosphates (PolyP2); 3, acid-insoluble polyphosphates (PolyP3,d,s);4, polyphosphate: ATP phosphotransferase; 5, 1,3-phosphoglycerate: polyphosphate phosphotransferase; 6, biosynthesis of polyphosphate connected with mannan synthesis; 7, biosynthesis of polyphosphate connected with nucleic acid synthesis; 8, tripolyphosphatase; 9, polyphosphatase; and 10, polyphosphate depolymerase.

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IGOR S. KULAEV AND VLADlMlR M. VAGABOV

the green sulphur bacterium Chlorobium thiosulphatophilium (Hughes et al., 1963; Cole and Hughes, 1965), the green alga Chlorella sp. (Iwamura and Kuwashima, 1964) and Acetabularia mediterranea (Rubtsov and Kulaev, 1977). In the last organism, a polyphosphatase hydrolysing highly polymeric polyphosphates to orthophosphate was also detected in the cell-free extract. In A. mediterranea, activities of both of the enzymes detected, polyphosphatase and polyphosphate kinase, increased markedly during growth of this alga under conditions of phosphorus starvation. C . METABOLISM OF POLYPHOSPHATES IN EUKARYOTES

As already mentioned, the metabolism and role of inorganic polyphosphates have been most completely studied in fungi and yeast. Therefore, the present section will be devoted to a detailed discussion of investigations conducted with these eukaryotic micro-organisms.

1. Yeasts and Fungi as Representatives of Heterotrophic Eukaryotic Micro-Organisms Yeasts are micro-organisms in which polyphosphates were not only first discovered (Liebennann, 1888) but in which their metabolism was also best studied (see reviews by Kulaev and Belozersky, 1962; Langen et al., 1962; Hoffmann-Ostenhof, 1962; Yoshida, 1962; Harold, 1966; Dawes and Senior, 1973; Matile, 1978; Kulaev, 1975, 1979; and articles by Weimberg, 1975; Ludwig et al., 1977; Diirr et al., 1979; Tijssen et al., 1980; Lusby and McLaughlin, 1980). Metabolism of polyphosphates has been fairly well studied in Neurospora crassa (Harold, 1966; Kulaev, 1979; Cramer et al., 1980; Trilisenko et al., 1980, 1982) and Aspergillus niger, Penicillium chrysogenum (Kulaev, 1979), Physarum polycephalum (Sauer et al., 1969; Goodman et al., 1969; Hildebrandt and Sauer, 1977; Sokolovsky and Kritsky, 1980), Dictyostelium discoideum (Gezelius, 1974; Al-Rayess et al., 1979) and in a number of parasitic fungi (Bennett and Scott, 1971; Wool and Held, 1976; Sysuev et a/., 1978; Umnov et al., 1981). Polyphosphate metabolism proved to be similar in all of the yeasts and fungi studied. Therefore, on the basis of all available data, it would be appropriate to attempt to draft a scheme of polyphosphate metabolism common to all these micro-organisms. Yet, early work on polyphosphate metabolism in fungi and yeasts (Kulaev and Belozersky, 1962; Langen et al., 1962; Harold, 1966; Kulaev, 1979) showed that a metabolic link existed

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

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between different fractions of polyphosphates having, as we now know, different chain lengths and dissimilar intracellular localization. Experiments with radioactive phosphate indicated that the high molecular-weight polyphosphates closest to the surface may be degraded, at particular stages of growth of these micro-organisms, to less polymerized fractions localized inside the cells. Thus, the possibility of transformations: PolyP+PolyPd+ PolyPpPolyP2+PolyPI was demonstrated. However, many questions remained unclear. For example, how do the most polymerized fractions of polyphosphates produce further less polymerized fractions? Are there independent pathways for synthesis and utilization of each of these fractions? In recent years it has become clear that specific fractions of polyphosphates represent different pools of these compounds characterized by specific metabolic features and related by their functions and biogenesis to particular cellular compartments (Kulaev, 1973a,b; Kulaev and Konoshenko, 1971a,b; Kulaev et al., 1972a,b; Konoshenko et al., 1973). Only one enzyme of polyphosphate metabolism (1,3-diphosphoglycerate:polyphosphate phosphotransferase) was found in the cytoplasmic volutin-like inclusions of N. crassa (Kulaev and Konoshenko, 1971a).Therefore, it may be concluded that metabolism of the Polyp, fractions localized in the cellular inclusions of N. crassa is very closely related to glycolysis. Formation and utilization of polyphosphates in these volutin-like granules may well be one of the mechanisms for regulating glycolysis in this fungus. We showed that the activity of this enzyme increased drastically in N. crmsa when in these fungal cells ATP synthesiswas inhibited by 8-aza-adenine(Kulaev et al., 1968).Thus, it may be suggested that polyphosphates of the plasmic volutin-like granules are most actively involved in the functioning and regulation of glycolysis under conditions when, in cells of N. crassa and possibly in other microorganisms, owing to some factors, metabolism of adenine nucleotides is blocked. Besides volutin-like granules, high molecular-weight polyphosphates were found in the nuclei of N. crassa (Kulaev et al., 1970d). They were also detected in similar structures of other yeasts and fungi (Skryabin et ul., 1973; Hildebrandt and Sauer, 1977; Sokolovsky and Kritsky, 1980). It is not yet understood how biosynthesis and utilization of polyphosphates are carried out in these cellular structures. Still, there is an extremely important phenomenon revealed in N . c r a m and other fungi and yeast (Kritsky et al., 1968, 1970; Melgunov and Kulaev, 1971; Kulaev et al., 1970d, 1973b, 1977). Direct correlation was established between rates of accumulation of nucleic acids, namely RNA, and of salt-soluble polyphosphates (PolyPz; Fig. 8) localized, at least partially, in the cellular nuclei. On the basis of these data, we suggested that, in nuclei, a mechanism is operative for the synthesis of polyphosphates from pyrophosphate formed during RNA synthesis with the help of RNA-polymerase (Fig. 9). Such a mechanism of inorganic polyphos-

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IGOR S. KULAEV AND VLADIMIR

0.3

-

M. VAGABOV

0.2 -

0.1

-

01. 0

e e

e e ee

I

0.f

I

0.2

Velocity of RNA synthesis

FIG. 8. Graph showing correlation between rates of formation of RNA and polyphosphate salt-soluble fraction PolyPz in Neurosporu crussa. From Kritsky et al. (1970).

phate (metaphosphate) formation was recently shown to operate in in uitro experiments on RNA biosynthesis with crude preparations of the DNAdependent RNA polymerase from E. coli (Volloch et al., 1979). Of evident interest are recent findings of Hildebrandt and Sauer (1977) who showed that, in nuclei of Physarum sp., polyphosphates are present in nucleoli, i.e. at the sites of rRNA synthesis and ribosome formation. They also revealed that in in vitro experiments this fraction is functioning as an inhibitor of RNA polymerase A which catalyses rRNA biosynthesis. Finally, in the process of differentiation of this fungus, the amount of this fraction of polyphosphates, referred to by the authors as “specific nucleolar initiation inhibitor”, varies strongly depending on the stage of differentiation. These results testify that polyphosphates may play an extremely important part in the life of organisms, regulating such an’important process as biosynthesis of nucleic acids. The paramount importance of this polyphosphate fraction for the function-

POLYPHOSPHATE METABOLISM I N MICRO-ORGANISMS

( n ) pyrophosphate

x

pyrophosphatase

(n

- I) pyrophosphate+orthophosphate

117

polyphosphate,

(7)

polyphosphate,

+I

FIG. 9. Proposed scheme for the interrelationship between biosynthesis of salt-soluble polyphosphatesand nucleic acids. From Kulaev er al. (1973b).

ing of any living cell is shown by the fact that they were found even in nuclei of higher animals (Penniall and Griffin, 1964; Bashirelashi and Dallam, 1970; Mansurova et af., 1975b; Offenbacher and Kline, 1980). In higher animals, high molecular-weight polyphosphates are found only in their nuclei. This indicates that polyphosphates are most important in these structures, being involved at all stages of development of living organisms. The third pool of polyphosphates occurring inside the plasma membrane is localized in vacuoles of fungal and yeast cells (Indge, 1968; Urech et af., 1978; Durr et af., 1979; Cramer et al., 1980; Okorokov et al., 1980; Lichko et al., 1982). Recently, numerous data have appeared pointing to the fact that polyphosphates having pronounced polyanionic properties participate in vacuoles primarily in the binding of considerable amounts of low molecular-weight compounds carrying a positive charge (Durr et al., 1979; Cramer et af., 1980; Allan and Miller, 1980; Okorokov et al., 1980; Lichko et al., 1982; Beckerich et af.,1981).According to the reports of Matile (1978), Durr et al. (1979), and Cramer et af. (1980), considerable amounts of arginine and lysine linked by ionic bonds to polyphosphates are accumulated in vacuoles of yeast and N. cassa. Generally, accumulation in vacuoles of these basic amino acids and polyphosphates in these organisms occurs simultaneously, though under certain extreme conditions they may be supplied to vacuoles separately. Another positively charged metabolite which accumulates in vacuoles and is bound in them to polyphosphates is S-adenosylmethionine (Allan and Miller, 1980). According to Okorokov et al. (1980) and Lichko et al. (1982), Mn2+and Mg2+may also accumulate in yeast vacuoles, being mostly bound

IGOR S. KULAEV AND VLADlMlR M. VAGABOV

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to polyphosphates. By binding cations, polyphosphates participate in regulating the turnover of these most important cellular metabolites. Of the enzymes of polyphosphate metabolism, vacuoles were found to contain polyphosphate kinase (Shabalin et ul., 1977) and a polyphosphate depolymerase which hydrolyses high molecular-weight polyphosphates to low molecular-weight fragments, possibly to trimetaphosphate (Schwencke, 1978;Durr et al., 1979). Table 5 shows that in whole cells of Succh. carlsbergensis, as in vacuoles, “reverse” polyphosphate kinase activity prevails catalysing ATP synthesis by transfer of the terminal phosphate from polyphosphates to ADP. Thus, vacuolar polyphosphate kinase may be actively involved in maintenance of a certain ATP level and, through this, of the contents of other nucleoside phosphates in yeast cells. As a result, polyphosphate kinase may take part in the utilization of polyphosphates stored in vacuoles during nucleic acid synthesis. The consumption of polyphosphates that are soluble in acids for synthesisof nucleic acids in fungi and yeast was emphasized in several reports. From Table 5 it is clear that, though the role of polyphosphate kinase is not significant in synthesis of polyphosphates from ATP in whole cells and protoplasts of yeast, in vacuoles of these organisms the activity of ATP: polyphosphate phosphotransferase increases markedly to bring about synthesis of polyphosphates. If this enzyme is localized in the tonoplast of vacuoles (Y. A. Shabalin, personal communication), it is possible that it plays a key role in symport through the membrane of phosphate and positively charged ions (Matile, 1978). Polyphosphate depolymerase may also prove to be important in metabolism of vacuolar polyphosphates. If this enzyme is localized in the tonoplast, then it may be assumed that it is also involved in metabolism and transport of cations and positively charged compounds into the yeast vacuoles (Matile, 1978). Kritsky and Chernysheva (1 973) suggested that polyphosphate depolymerTABLE 5. Activity of polyphosphate kinases in some subcellular fractions of Saccharomyces carlsbergensis. After Vagabov and Shabalin (1 979) Activity (mE (mg protein)-’) Fraction Vacuoles

Cell envelope

Protoplast lysate

ATP: polyphosphate phosphotransferase

Polyphosphate: ADP phosphotransferase

1.24

4.10

0.053 0.033

E indicates an International Unit of activity.

4.20 3.40

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

119

ase may participate not only in breakdown of highly polymerized polyphosphates to less polymerized ones, but also in translocation of the fragments formed through membranes at the expense of the energy released during cleavage of phosphoanhydride bonds. If this suggestion is correct then, during the translocation of short-chain polyphosphate, symport of positively charged ions and metabolites may occur. The interrelations between accumulation in yeast cells of short-chain polyphosphates and positively charged amino acids were investigated by Ludwig et al. (1977) and Lusby and McLaughlin (1980). These authors showed that addition of free L-amino acids, particularly arginine and lysine, to yeast culture media under conditions of nitrogen starvation resulted in an intensification of yeast growth and rapid intracellular accumulation first of tripolyphosphate and then of more polymerized chains, including tetrapolyphosphate and pentapolyphosphate. Experimentswith 32Pshowed that the tripolyphosphate formed originated not from orthophosphate supplied in the medium, but from a more polymerized polyphosphate fraction. Accumulation of tripolyphosphate and, to a lesser extent, of other smaller polyphosphates in cells of intensively growing yeast was also observed by Solimene et ul. (1980). These results are very interesting, although unfortunately they do not provide sufficient information about the localization of low molecular-weight polyphosphates in yeast cells during intensive growth. Here, one undoubtedly deals with the Polyp, fraction which accumulatesin yeast cells either in vacuoles or in the cytoplasm. Low molecular-weight polyphosphates accumulate in the cytoplasmic(vacuolar) granules of yeast (at least under conditions of an intensive L-amino acids uptake) as a complex with positively charged arginine and lysine residues. Such electroneutral complexes represent a pool of negatively charged phosphate and positively charged amino acids in a rather inert form convenient for the cell. The polyphosphate complex with arginine (or lysine) which accumulates in vacuoles and probably to some extent in the yeast cytoplasm, resembles cyanophycin, which is a copolymer of aspartic acid and arginine discovered by Simon (1971) in blue-green algae. This copolymer, in which the role of a negatively charged complex is performed by aspartic acid, accumulatesin cells of blue-green algae as granules and forms an intracellular reserve of the most important nitrogen-containing metabolites. Recalling the investigations of McLaughlin and his coworkers, one should take into account the formation of tripolyphosphate in yeast cells from high molecular-weight polyphosphate fractions in the presence of supplied L-amino acids. It is difficult to state which polyphosphate fraction is depolymerized to tripolyphosphate. It should be noted that polyphosphate depolymerase was found in cells of N. crussa (Kulaev et al., 1972a) both outside the cytoplasmic membrane

120

IGOR S. KULAEV AND VLADlMlR M . VAGABOV

TABLE 6. Intracellular localization of polyphosphate depolyrnerase activity in Neurosporu crussu. After Kulaev et ul. (1 972a) ~~

Activity (mE(mg protein-’)) Substrate

Intact cells

Protoplasts

Polyphosphate (ii=290) Polyphosphate (ii= 180)

8.3 9.4

0.6 1.3

Nuclei

Mitochondria

0.15

0.20

0.0 0.0

Microsomes Cytosol 2.2

-

0.0 0.0

E indicates an International Unit of activity and ri is chain length.

(major part) and in nuclei and the “microsomal” fraction into which vacuoles were undoubtedly fragmented during fractionation (Table 6). Schwencke (1 978) also detected polyphosphate depolymerase directly in yeast vacuoles. Its action on high molecular-weightpolyphosphates yielded tripolyphosphate as the final product. It is not precluded that, in the presence of L-amino acids, the depolymerase contained in nuclei hydrolyses high molecular-weight polyphosphate bound in nucleoli to RNA polymerase A thereby inhibiting this enzyme, thus contributing to RNA and protein synthesis, as well as culture growth. Tripolyphosphate formed during depolymerization may be a “primer” for synthesizing“de novo” high molecular-weight polyphosphates. It appears quite probable that tripolyphosphate may be the form in which polyphosphates could be translocated through the cellular membranes. This suggestion correlates well with the finding of Valikhanov and Sagdullaev (1979) indicating that tripolyphosphate uptake from the medium by cotton roots is more rapid compared with other phosphates, including orthophosphate. Thus, it appears probable at present that, in fungal cells and possibly in cells of other organisms, tripolyphosphate is, on the one hand, the form of polyphosphate appropriate for transport through membranes and, on the other hand, a “primer” of the biosynthesis “de novo” of high molecularweight polyphosphates. It appears probable that the repeatedly shown (Kulaev, 1979) reversible transformations: PolyPz$PolyP1 are connected with the existence in cells of fungi and yeast of the system:

-

High molecular-weight polyphosphates tripolyphosphate

polyphosphate synthases

plyphosphate

depo1ym erasc

high molecular-weight polyphosphates

Such transformations were clearly demonstrated recently during yeast dehydration followed by their reactivation (Kulaev et a/., 1977; Table 7).

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POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

Summarizing about the metabolism of polyphosphate fractions localized in cells of yeast and fungi inside the cytoplasmic membrane (cytosol, vacuoles, nuclei), it is appropriate to underline their multifunctional roles. They are not only reservoirs of phosphates and energy, but also most important regulators of cellular metabolism. They participate in the regulation of ATP, ADP, ortho- and pyrophosphate levels, in control of glycolysis and intracellular ionic fluxes and, finally, in regulation of nucleic metabolism and growth processes in general. TABLE 7. Contents (pg P (g dry wt cells)-') of orthophosphate and polyphosphate fractions of Succhuromyces cereuisiue-14 grown in a molasses medium in a fennentor then dehydrated or reactivated. After Kulaev et ul. (1977) Content (pg P (g dry wt cells)-') in Phosphorus compound of fraction

Initial cells

Cells dehydrated at 37°C for 24 hours

Cells reactivated at 37°C for 30 minutes

Orthophosphate Acid-soluble (PolyP,) Salt-soluble (PolyPz) Alkali-soluble (PolyPs) Hot perchlorate extract (PolyPs) High molecular-weight polyphosphates (total)

2150 350 4470 1340 1160

1830 3710 850 1570 210

2980 620 1910 1530 520

7230

6340

4580

Significant amounts of the most polymerized polyphosphates are localized

in fungi at the cell periphery, close to the cytoplasmic membrane (Weimberg

and Orton, 1965; Kulaev et al., 1966, 1967a, 1970a,b; Souzu, 1967a,b; Weimberg, 1970; Vagabov et al., 1973). Studies on localization of polyphosphate metabolism enzymes in N.c r a m revealed that the cell periphery, in the proximity of the pool of polyphosphates, contains substantial amounts of polyphosphate depolymerase which hydrolyses polyphosphates in the middle of the chain (Kulaev et al., 1972a) and polyphosphatase splitting off terminal phosphate residues (Kulaev et al., 1972b;Trilisenko et al., 1980). Recently, Trilisenko et al. (1980,1982)isolated a mutant of N . crassa with a markedly low polyphosphatase activity. In this mutant, hydrolysis of high molecular-weight polyphosphates (i= 180) by polyphosphatase proceeded at a lower rate than with the same enzyme from the wild-type strain of N. crassa. The affinity of this polyphosphatase for high 180) proved to be two orders of molecular-weight polyphosphate (i= 9). magnitude higher compared to low molecular-weight polyphosphate (i=

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IGOR S. KULAEV AND VLADlMlR M. VAGABOV

This observation and accumulation of highly polymerized polyphosphates in the mutant with low polyphosphatase activity (Table 3) argue in favour of the participation of this enzyme in uivo in utilization of polyphosphates with a peripheral location in fungal cells. Data on the peripheral localization of highly polymerized polyphosphates were also obtained for Endomyces magnusii (Kulaev et al., 1967a;Kulaev, 1979), Sacch. carlsbergensis (Vagabov et al., 1973) and Sacch. cereuisiae (Tijssen et al., 1980). It should be stressed that, in N . crassa, polyphosphatases degrading polyphosphates at terminal phosphate residues are apparently firmly bound to the cytoplasmic membrane. This conclusion may be drawn from the fact this enzyme is removed from the protoplast surface only after treatment with the detergent Triton X-100(Kulaev ez al., 1972b;Kulaev, 1973b;Konoshenko et al., 1973; Krasheninnikov et al., 1973). Some indirect data are available (Kulaev, 1979) which indicate that high molecular-weight polyphosphates in the peripheral part of the cell are localized, in N . crassa and E. magnusii,close to the polyphosphatase hydrolysing them to orthophosphate, i.e. in the proximity of the cytoplasmic membrane. Under various conditions affecting the cytoplasmic membrane of these organisms, these are the fractions of highly polymerized polyphosphates that are subject to hydrolysisto form orthophosphate. Unsuccessful attempt of Wiemken and his co-workers (Diirr et al., 1979) and Davis and his coworkers (Cramer et al., 1980) to detect peripheral fractions of polyphosphates in yeast and N. crassa cells were, beyond any doubts, due to this cause. The methods they used for pretreatment and fractionation of cells caused a prompt and selective breakdown to orthophosphate of very highly polymerized fractions of polyphosphates localized in the cytoplasmic membrane near to polyphosphatase. The latter enzyme, and the above-mentioned polyphosphate depolymerase whose occurrence in yeast and fungi was reported in a number of studies (Malmgren, 1949, 1952; Mattenheimer, 1951; Kritsky et al., 1972), are responsible in fungi for utilization and degradation of the most polymerized polyphosphate fractions (PolyP3, Polyp4 and PolyPs). It seems that, in yeast and fungi, the depolymerase functions in transformation of highly polymerized polyphosphates localized outside the plasma membrane giving rise to less polymerized polyphosphates capable of being translocated through the ‘membrane (Belozersky and Kulaev, 1957; Kulaev et al., 1959; Langen et al., 1962; Kritsky and Chernysheva, 1973), while the polyphosphatase has quite a different function in metabolism of these compounds. Over the past 15 years, Van Steveninck, in collaboration with other Dutch researchers, has obtained convincing results pointing to the fact that highly polymerized polyphosphates, localized at the periphery of yeast cells, are involved as energy donors in the basic transport of sugars through the cytoplasmic membrane (Van Steveninck, 1963; Van Steveninck and Booij,

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1964; Dierkauf and Booij, 1968; Jaspers and Van Steveninck, 1975; Tijssen et al., 1980). Umnov et al. (1974a) and Trilisenko et al. (1980) showed that polyphosphatase plays an important role in utilization of these polyphosphate fractions for active transport of sugars through the cytoplasmic membrane in N. cra~saby hydrolysing them to orthophosphate. It is admitted that polyphosphatase may operate in vivo in yeast and fungi not only as a phosphohydrolase but also as a phosphotransferase. It may also transport activated phosphoryl derivatives of polyphosphates instead of water to some components of the system for active sugar transport, acting as energy donor for this process. The ability of some phosphohydrolases to carry out certain phosphotransferase reactions has been known for a long time (Nordlie and Arion, 1964; Stetten, 1964). Returning to investigations of localization of enzymes of polyphosphate metabolism in fungi, it should be noted that, in the region of localization of the most polymerized polyphosphates in the proximity of the cytoplasmic membrane, we detected only enzymes of degradation and utilization of polyphosphates. Until recently it was not known how polyphosphates of this fraction are synthesized. Certain progress in solving this problem has been achieved in our laboratory in the course of investigation of polyphosphate metabolism in yeast (Kulaev et al., 1972c,d; Vagabov et al., 1973; Tsiomenko et al., 1974a,b; Vagabov and Shabalin, 1979; Shabalin et al., 1978, 1979). These reports showed good correlation (Table 8) between rates of accumulation of polymerized polyphosphates localized in the cellular envelope and synthesis of polysaccharides of the cell wall (Kulaev et al., 1972c,d). The highest value for the correlation coefficient (0.8-0.9) was found for the Polyp4

TABLE 8. Correlation coefficients between the rates of formation of various polyphosphate fractions and polysaccharides in Saccharomyces carlsbergensk After Kulaev et al. (1972c,d) Polysaccharides

Polyphosphates

Correlation coefficients"

(Polysaccharides) Glycogen Glycogen Glycogen Glucan + mannan Glucan Glucan Mannan Mannan Mannan

(Polyphosphates) PolyP, PolyP2 (PolyP2, PolyP3, PolyP4, PolyPs) (PolyP2, PolyP3, PolyP4, PolyPs) PolyPz PolyP3 PolyP2 PolyP3 PolyP4

0.806f 0.068 0.077f 0.02 0.141 f 0.008 0.173f0.018 0.750f 0.087 0.291k0.180 0.615f0.122 0.136f 0.192 0.035f 0.196 0.813 0.098

The coefficients were calculated from the results of 36 determinations.

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fraction and mannan synthesis. The behaviour of these two fractions during normal growth, as well as during various impositions on culture growth, was a clear-cut parallelism. This can be seen from Fig. 10. These data suggested the existence of some specific interrelation between metabolism of these two compounds which, though quite different in their chemical nature, are nevertheless components of the same organelle, namely the cell envelope. Synthesis of mannoproteins from GDP-mannose is known to proceed not in the envelope itself but inside the cell, in the so-called microsomal fraction, and in vesicular membrane structures which bud off to the localization site in the cellular envelope(Matile, 1975; Lehle et al., 1977; FarkaS, 1979; Schekman et al., 1981). Experiments with [P-32P]GDP-['4C]mannose and the microsomal fraction of Sacch. carlsbergensis showed that GDP-mannose is not only the donqr of mannosyl groups during biosynthesisof mannoproteins (Brehrens and Cabib, 1968), but also acts as the source of phosphate in biosynthesis of polyphosphates (Shabalin et al., 1978; Vagabov and Shabalin, 1979; Kulaev et al., 1979). It was also established that syntheses of both mannan and polyphosphates require Mn2+, whereas Mg2+ inhibited both processes. Further investigations indicated that transport of phosphate groups from GDP-

I2O

c

loot

c

c

a3

c

c

0

u

50

60

70 00

Time (hours)

FIG. 10. Changes in the contents of mannan (0)and high molecular-weight polyphosphate fraction Polyp4 ( 0 ) under different growth conditions of Saccharomyces carlsbergensis in Rider medium in the presence (a) or absence (b) of nitrogen. From Kulaev et al. (1972c,d).

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mannose to polyphosphates involves a lipid intermediate identical in its characteristics to dolichol-pyrophosphate mannose (Vagabov and Shabalin, 1979; Shabalin et al., 1979). On the basis of the results obtained, the mechanism of biosynthesis of polyphosphates from GDP-mannose in yeast Sacch. carlsbergensis may be depicted as follows:

+

+

1. GDP-mannose dolichol phosphate4 GMP dolichol-pyrophosphatemannose 2. Dolichol-pyrophosphatemannose (mannan).+dolichol pyrophosphate (maman),,+I 3. Dolichol pyrophosphate +@olyphosphate)n-rdolicholphosphate+ @olyphosphate)n+I

+

+

Available data lead us to conclude that, in yeast, a new pathway for biosynthesis of high molecular-weight polyphosphates has been shown to be closely related to the synthesisof mannoproteins of the cell wall. Thus, it stems from the above data that both biosynthesis and utilization of the surfacelocated highly polymerized polyphosphates are closely connected with the biogenesis and functioning of a most important cellular compartment of yeast and fungi, namely their cellular envelope. It is noteworthy that not only biosynthesis but also degradation of polyphosphates and mannoproteins of yeast are closely interrelated. As shown in Fig. 11, practically synchronous changes in polyphosphatase and mannosidase activities occur during yeast cultivation (Tsiomenko et al., 1974a,b). Metabolism of these two biopolymers of the cellular envelopes seems to be closely co-ordinated. Data pointing to a relation between biogenesis and functioning of a polyphosphate fraction and that of the fungal cell wall were also obtained by Wool and Held (1976). Using ultracytochemical and X-ray dispersion analyses, these authors studied localization of polyphosphates in isolated zoospores of Rozella allomycis fungus parasitizing species of Allomyces. In this work, vesicles of the endoplasmic reticulum were shown to contain both polysaccharide precursors of the cyst cell wall and polyphosphates; in cysts, polyphosphates were found in the regions of the cytoplasmic membrane and cell wall. In this fungus, in addition to the above sites, a polyphosphate fraction was detected in vacuoles of cysts before germination. The authors believe that formation of these polyphosphates was connected with degradation of a nucleic acid fraction, whereas their utilization (hydrolysis to orthophosphate) occurred immediately before the start of cyst germination, thus creating the required osmotic pressure for the ‘‘explosion’’ of cysts and penetration of germ cells into the tissue of the host fungus. It should be recalled that in earlier literature on polyphosphates, similar mechanisms for production of specific polyphosphate fractions (during degradation of nucleic acids) as well as those of their utilization (for creating excessive osmotic pressure) were frequently demonstrated and discussed (Harold, 1966; Kulaev, 1979). In particular, Kritsky and his colleagues showed that the osmotic pressure developed during hydrolysis of

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IGOR S. KULAEV AND VLADlMlR M. VAGABOV

-8

0.6

-6

0*40>L9 0 0.04

0.2

0

-

0

1

0

I

0

I

I

3

0.02

I

0

5

Time (hours)

FIG. 11. Variation in the activity of enzymes hydrolysing polyphosphates (a) and mannan (b) in Succharomyces carlsbergensis after their transfer into fresh medium. 0, Activities of polyphosphatase and a-mannosidase in (a) and (b) respectively; 0 , content of polyphosphate and mannan, in (a) and (b) respectively. E indicates one International Unit of Activity. From Tsiomenko et ul. (1974a,b).

polyphosphates in the lamellae of fruiting bodies of Agaricus bisporus is involved in dissemination of spores (Kulaev et al., 1960; Kritsky et al., 1965a,b). Data from Gezelius (1974) also argue in favour of the existence of quite different pathways for polyphosphate formation in various fungi. During investigation of polyphosphate metabolism in Dictyostelium discoideum, Gezelius showed that large amounts of polyphosphates were synthesized during the transition of D . discoideum from the amoeboid to the aggregated stage. Dictyostelium discoideum is known to produce cyclic AMP intensively

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from ATP during this period (Pastan et al., 1975). It may be that this fungus possesses an enzyme system that catalyses synthesis of polyphosphates from pyrophosphate formed during biosynthesis of cyclic AMP from ATP. Gezelius (1974) also pointed to the existence in this fungus of a mechanism for polyphosphate formation different from its synthesis with the participation of polyphosphate kinase. The occurrence of several mechanisms of polyphosphate synthesis was also confirmed genetically. Beckerich et al. (1981) isolated a number of mutants of Saccharomycopsis lipolytica (Table 9) which lacked certain fractions of TABLE 9. Distribution of polyphosphate fractions in mutants of Saccharomycopsis lipolytica. After Beckerich et al. ( 1981)

Polyphosphate fraction (nmol K2HP04 equiv. (mg dry wt)-l) Strain

FI

F2

15901.7

0

33

PlY 1 PlY 2 PlY 3 ply 4 ply 5

2 8 0 10 25

PlY 7 PlY 9

0

0

F3

F4

Fs

7

0 0 4 0 0 0 0 0 0

14 0 0 0 0

5 0 5

0

3 5 0 4

0 0

5

1 0

0

7

6

0 0 0 1

FI indicates an acid-solublepolyphosphate(ri= I-5),F2 a perchlorate-soluble polyphosphate (R up to 20), F3 a polyphosphate with ii=20-50, F5 a polyphosphate with A= 50-250 and F4 nucleic acids.

acid-insoluble polyphosphates, while the PolyPl/PolyP2ratio differed greatly from that in the parental strain. It was suggested that, in the mutants, the pathway for polyphosphate biosynthesis related to formation of the fungal cell wall was impaired. That the acid-insoluble polyphosphate fractions are involved in formation of the cell wall is supported not only by these results (Vagabov and Shabalin, 1979; Shabalin et al., 1979) but also by the data of Sokolovsky and Kritsky (1980) and those of Trilisenko et al. (1982) on the absence of these very fractions from Physarum polycephalum and a slime mutant of N. crassa (Fig. 12).

IGOR S. KULAEV AND VLADlMlR M. VAGABOV

128

Strain ad-6

f -

4-:"1.5

o o r L.b:C

0 10 20 30

Slime mutant

30,lS-3

L/ o:;v 1: 0

15

30

50

-.A=&

0 10 20 30

0

Time (hours)

FIG. 12. Time-course of changes in the contents of various polyphosphate fractions during growth of Neurospora crassa: strain ad-6; a leaky mutant in polyphosphatase (30,19-3) and a slime mutant devoid of the cell envelope. 0 Indicates the growth phase of N . crassa and 0 indicates phosphate content in the polyphosphate fraction.

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2. Algae as Representatives of Phototrophic Eukaryotes The autotrophic nature of algae affects their polyphosphate metabolism (Miyachi et al., 1964; Kulaev and Vagabov, 1967;Ullrich and Simonis, 1969; Kuhl, 1974). In numerous reports it was shown that formation of polyphosphates and polyphosphate granules in algae occurs with a markedly higher intensity in the light than in the dark (see Kulaev, 1979). The most fundamental and detailed investigation of polyphosphate metabolism in algae was conducted by Miyachi et al. (1964) with Chlorella ellipsoidea. They showed that only one of four polyphosphate fractions detected in C. ellipsoidea was formed in the light. Both biosynthesis and utilization of polyphosphates were shown to occur in the light. It was also established that, in C. ellipsoidea as in heterotrophs, the fraction of polyphosphates extracted with cold acid was a component of volutin. According to Atkinson et al. (1974), in C . ellipsoidea these granules may be localized, at least partially, in vacuoles. Accumulation of polyphosphates contained in these granules also depends, according to Miyachi et al. (1964), on photosynthesis, since they are formed from the fraction synthesized in the light. The biosynthesis and utilization of two other fractions detected by Miyachi et al. (1964) in C. ellipsoidea were totally independent of photosynthesis. Their metabolism depended on the presence of phosphate in the incubation medium. Similar results were obtained later by Kanai and Simonis (1968) for Ankistrodesmus braunii. It is interesting that, in these and other early studies of polyphosphate metabolism in algae, a close and fairly complex relationship between certain polyphosphate fractions and nucleic and metabolism was established and found to be similar to that observed in heterotrophic organisms (Kulaev, 1979). Bearing in mind the data of Richter (1966), who demonstrated polyphosphate synthesisin the nucleus-freecell halves of Acetabularia sp., one may draw an indirect conclusion that the presence of the cell nucleus is not obligatory for the replenishment of at least some polyphosphate fractions in algae. In general, studies on polyphosphate metabolism in this gigantic unicellular alga appear to be very promising. In our laboratory, for example, Rubtsov and his coworkers (Kulaev et al., 1975)found that in the early stages of growth of Acetabularia mediterranea and Acetabularia crenulata, in contrast to heterotrophic organisms, only acid-soluble (Polyp,) and salt-soluble(PolyPz) fractions-were present (Table 10). At the stage af cyst formation (stage 4), characterized by intensive synthesis of their cell wall components, the distribution of these compounds in fractions in Acetabularia cells does not differ qualitatively from that in heterotrophs. At this stage of growth, in

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

TABLE 10. Contents of inorganic polyphosphates and orthophosphate in Acetabularia crenulata at different stages of development. After Kulaev et al. (1975) Phosphate content &g phosphorus cell-I) Fraction Orthophosphate Acid-soluble (PolyP~) Salt-soluble (PolyP2) Alkali-soluble (PolyPs) Hot perchloric acid extract (PolyP~) Total polyphosphates

Stages of culture growth: 1 2 3 4 0.52 0.67 0.12 0.0

1.40 3.30 0.46 0.0

0.76 1.88 10.10 2.41 0.44 1.27 0.25 0.54

0.79

0.0 3.76

0.0 10.79

0.81 5.03

Stages of growth were as follows: 1, young cells 1.5-2 cm long; 2, cells 2.5-3.0 cm long, up to 2 mm in diameter; 3, cells with umbellulles filled with secondary nuclei; 4, cells with mature umbellulles filled with cysts.

addition to acid- and salt-soluble polyphosphates (as in active young photosynthetic cells), alkali-soluble (Polyp3 and PolyP4) polyphosphates and those extractable by hot perchloric acid appear. In studies with A. mediterranea, it was first shown that high molecular-weight polyphosphates do not occur in chloroplasts (Rubtsov et al., 1977). Structures connected with photophosphorylation could not be shown to be capable of polyphosphate biosynthesis. The absence of high molecular-weight polyphosphates from chloroplasts was also confirmed for higher plants such as cotton (Valikhanov and Sagdullaev, 1979). However, as shown on p. 149, light-dependent synthesis of inorganic pyrophosphate attended by electron transfer along the electron-transport chain was revealed in chloroplasts of A. mediterranea and pea (Rubtsov et al., 1976). Investigations of A. mediterrunea provided the answer to the question of whether light-dependent accumulation of polyphosphates in algae was directly connected with photosynthesis itself or whether their formation in the light was simply stimulated by increased synthesis of ATP or pyrophosphate at the expense of photosynthetic phosphorylation. Detection of ATP: polyphosphate phosphotransferase in A. mediterranea (Rubtsov and Kulaev, 1977), as well as the absence of high molecular-weight polyphosphates and their biosynthesis in chloroplasts of this alga, together with corresponding inhibitor analysis, point convincingly to the fact that high

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molecular-weight polyphosphates are not directly, but rather indirectly, connected with photosynthesis through formation of ATP (but not of pyrophosphate) in photosynthetic phosphorylation. Taking into account that, in algae, polyphosphates accumulate in the light essentially in vacuoles (Atkinson et af.,1974; Sundberg and Nilshammar-Holmvall, 1979, together with detection of polyphosphate hydrolase activity in A. mediterranea (Rubtsov and Kulaev, 1977), one can depict the basic metabolic pathways of the light-dependent synthesis and utilization of polyphosphates in algae as in Fig. 13. According to this scheme, by analogy with yeast (Shabalin et al., 1977), polyphosphate kinase is localized in vacuoles. However, this assumption still requires experimental support. When reviewing studies on polyphosphate metabolism in algae reported

light

FIG. 13. Major metabolic pathways for light-dependent synthesis and utilization of polyphosphates (polyp) in algae.

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

during the past years, reference should be made to the work of Peverly et al. (1978) and Adamec et al. (1979). These authors studied the influence of potassium ions on accumulation of polyphosphate granules in Chlorella pyrenoidosa. Peverly et al. (1978) found that K + ions stimulate polyphosphate granule formation in this alga. These authors also showed a correlation between accumulation of phosphate and potassium in cells which, after phosphorus starvation, were transferred to a medium containing adequate amounts of both components. Microscopic examination revealed intensive accumulation of polyphosphate granules in cells. Further, using X-ray dispersion analysis in combination with electron microscopy, Adamec et al. (1979) detected potassium in addition to phosphorus in polyphosphate granules. On the basis of these results, these authors believe that K + is the major cation of polyphosphate granules in Chlorellapyrenoidosa growing in a medium with a sufficient amount of potassium. D. NEW DATA O N POLYPHOSPHATE METABOLISM IN PROKARYOTES

At the present time, polyphosphate metabolism in prokaryotic microorganisms has been most extensively studied in mycobacteria (Winder and Denneny, 1957; Mudd et al., 1958; Drews, 1962; Dirheimer, 1964; Szymona and Ostrowski, 1964), corynebacteria (Sall et al., 1958; Hughes and Muhammed, 1962),propionic-acid bacteria (Kulaev et al., 1973a),streptomycetes (Kulaev et al., 1976), Aerobacter aerogenes (Harold, 1966) and Escherichia coli (Nesmeyanova et al., 1973a, 1974a). Detailed information about the characteristic features of the metabolism of these and a number of other eubacteria can be found in Kulaev’s (1979) monograph. The most important and experimentally supported inference from this work was the close relationship between polyphosphate and nucleic acid metabolism. We have postulated one possible mechanism for the interrelation between these two pathways (Kulaev et al., 1973b; Kulaev, 1975). The suggestion, based on our own and literature data, was that conjugation of these two metabolic pathways may occur at the level of synthesis of specific polyphosphate fractions from pyrophosphate formed during biosynthesis of nucleic acids. Recently Volloch et al. (1 979) in Tummerman’s laboratory confirmed this experimentally using a crude preparation of DNA-dependent RNA polymerase and phage SV-40 as a DNA template in in vitro experiments. They found that pyrophosphate (PP) formed during RNA biosynthesis by the preparation was not accumulated as such but condensed to form some polymeric phosphorus compound. For some unclear reasons, this compound was termed “trimetaphosphate”, though this work provided no valid support for such a term. It is interesting that use of purified preparations of

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DNA-dependent RNA polymerase and highly purified preparations of DNA polymerase I enabled the authors to observe formation of only pyrophosphate. These data prompt one to think that, in such a system, the synthesis of polyphosphate from pyrophosphate is not carried out by RNA and DNA polymerases but by a pyrophosphate-polyphosphate phosphotransferase tightly coupled to them. In recent years, a series of cytological data obtained with cyanobacteria and using electron microscopy have appeared. For these prokaryotes, the above-mentioned results demonstrated a very close topological relation between polyphosphates and nucleic acids. The most detailed information in this respect was provided by research from Jensen’s laboratory (Jensen, 1968, 1969; Jensen and Sicko, 1974; Sicko-Goad and Jensen, 1976; Sicko-Goad et al., 1975,1978;Lawry and Jensen, 1979;Baxter and Jensen, 1980a,b)as well as from a number of other laboratories (Kessel, 1977; Vaillancourt et al., 1978; Barlow et al., 1979; Ferguson et al., 1979) engaged on studies of phosphate metabolism in cyanobacteria. It should be noted that, at present, investigationof phosphorus metabolism in cyanobacteria is a very urgent problem. This is due to the “fluorescence” of cyanobacteria in inland water bodies which receive large amounts of various detergents and other phosphate-containing effluents of industrial production. In detergents, the most frequently used compound is sodium tripolyphosphate. Inland water bodies, particularly those located in industrialized countries, normally contain low concentrations of phosphorus (about 10 pg 1-’ or less) and suffer a massive “fluorescence” of cyanobacteria when substantial quantities of tripolyphosphate are “dumped” into them in waste waters. Conditions are created known as “phosphate overplus”. After a long phosphorus starvation, microbes finding themselves in a phosphorus-rich medium start to grow and reproduce very intensively. Under such conditions, cyanobacteria and other micro-organisms (Drews, 1962; Harold, 1966; Kulaev, 1979) accumulate large amounts of polyphosphates essentially localized in polyphosphate granules. The above authors studied in detail intracellular localization and chemical composition of polyphosphate granules under conditions of normal growth as well as under a lack or excess of certain nutrients in the medium. Investigating localization of polyphosphates in Plectonema boryanum, Jensen (Jensen, 1969; Jensen and Sicko, 1974) found that they were localized in this blue-green alga in five cellular sites. These were sites of ribosome formation, DNA fibrils, near thylakoids, polyhedral bodies which harbour key enzyme of photosynthesis, including ribulose 1$diphosphate carboxylase, and the nucleoplasmic zone. This implies that accumulation of polyphosphates in P. boryanum is basically conditioned by photosynthesisas well as biosynthesis and degradation of nucleic acids. It is interesting that in

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IGOR S. KULAEV A N D VLADlMlR M. VAGABOV

the first study devoted to polyphosphate formation in P . boryanum, Jensen (1969) reported that these compounds are detected cytochemically inside cell walls. Generally, in cyanobacteria, intracellular localization of polyphosphates does not differ from that in eukaryotic micro-organisms, particularly in phototrophs. Therefore, it can be concluded that their metabolism in cyanobacteria is primarily connected with nucleic acids and cell wall components and, to some extent, with functioning of the photosynthetic apparatus. It is not clear so far whether, in cyanobacteria, polyphosphates are produced directly by photophosphorylation or are secondary products from ATP. Shady et al. (1976) found that, in the phototrophic bacterium Rhodospirillum rubrum, polyphosphates are formed in chromatophores in the light indirectly, via ATP with participation of polyphosphate kinase. The availability of polyphosphate kinase in cyanobacteria, in particular in Anacystis nidulans, was demonstrated by Vaillancourt et al. (1978) who isolated “leaky” mutants in this enzyme. Of interest also is the fact that these mutants did not have polyphosphate granules observable by electron microscopy. It may be inferred that, in blue-green algae, polyphosphate kinase plays a very important part in phosphate metabolism. In this respect, A. nidulans is apparently similar to Aerobacter aerogenes mutants which are deficient in polyphosphate kinase and are also devoid of the ability to accumulate polyphosphates. It is noteworthy that, in A. nidulans, judging from the data of Vaillancourt et al. (1978), polyphosphatase has not been detected. Failure to detect this enzyme is very rare in micro-organisms. Ferguson et al. (1979) carried out an interesting study of mechanisms of polyphosphate synthesis in Paracoccus denitrijicans using 31Pnuclear magnetic resonance. Using inhibitors of oxidative phosphorylation and different conditions of energy metabolism, the authors showed the extreme importance of polyphosphate metabolism in the energy metabolism of this bacterium. In contrast to Harold (1966), the authors drew a conclusion shared by most researchers (see Kulaev, 1979), namely that, in bacteria, polyphosphates are reserves not only of phosphates but of energy also. In this work, it was observed that polyphosphates Polyp,, though synthesized in P . denitrijicans at the expense of the energy of succinate oxidation (possibly via intermediate ATP formation), do not utilize orthophosphate as phosphate source but use some intracellular phosphorus-containing compounds (possibly nucleic acids or products of their degradation such as nucleoside monophosphates). In recent years, some results were reported on mechanisms of polyphosphate utilization in bacteria. Butukhanov et al. (1979) reported intensive ATP synthesis (0.6-1.0 mg (ml medium)-’) from exogenous adenine in autolysing cultures of Corynebacterium sp. VSTII-301.They also reported data indicating that high molecular-weight polyphosphates and inorganic pyrophosphate

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were phosphate donors for ATP synthesis. It was calculated that, during the 24 hour growth of the autolysing cells of this culture, 58.3 pmol of acid-labile phosphate of ATP and ADP were synthesized and, at the same time, 33.5 pmol of acid-labile phosphate of intracellular polyphosphates and pyrophosphate were utilized. The author succeded also in isolating and purifying from culture not only polyphosphate: ADP phosphotransferase but also a new enzyme, pyrophosphate: ADP phosphotransferase. Moreover, Butukhanov et al. (1979) revealed that, after addition of exogenous adenine to a culture of Corynebacterium sp. autolysed for 72 hours, the activity of polyphosphatase activity decreased and that of polyphosphate: ADP phosphotransferase increased. After 84 hours when most of the adenine added was utilized for synthesis of ATP, polyphosphatase activity increased again in cells. This work demonstrated a competitive relationship between two enzymes, namely polyphosphate kinase and polyphosphatase, involved in utilization of polyphosphates. Similar competitive relationships between two different polyphosphate-utilizing enzymes were reported by Ziizina et al. (1981). In this work, results were obtained supporting previous observations (Kulaev et al., 1976; HoStalek et al., 1976; Kulaev, 1979) that, during antibiotic production in prokaryotes, inorganic polyphosphates, but not ATP, are used as an energy source. Polyphosphate utilization during synthesis of the antibiotic levorin was brought to light by Ziizina et al. (1981). They showed that polyphosphate utilization proceeds under conditions of phosphorus starvation with the help of polyphosphate glucokinase. As seen from Fig. 14, enzyme activity is dramatically enhanced in a culture of Streptomyces levoris producing this antibiotic under conditions of phosphorus starvation, and its variation clearly correlates with levorin accumulation. At the end of the stationary phase of growth of Strep. levoris, polyphosphatase activity increased, whereas polyphosphate glucokinase activity decreased. Substitution of the enzymes of polyphosphate metabolism may possibly be due to a deceleration of antibiotic formation during this period. In connection with this work, which demonstrated an important physiological role for glucose phosphorylation at the expense of high molecular-weight polyphosphates, recent research carried out in Szymona’s laboratory should be cited (Szymona et al., 1977; Szymona and Szymona, 1978,1979; Pastuszak and Szymona, 1980) dealing with structure and function of an enzyme catalysing this process in Nocardia sp. and mycobacteria, micro-organisms closely related to streptomycetes. At present, the specificity and individual features of this enzyme remain unclear. Returning to the problem of the possible functions of high molecularweight polyphosphates in prokaryotes, it should be noted that the most important role for these compounds is the regulation of orthophosphate level

136

IGOR S. KULAEV AND VLADlMlR

M. VAGABOV

r

-

0 D

v)

c .-

c

5

-P

m

0 1

3

6

9

Time (days)

FIG. 14. Correlation between the activity of polyphosphate glucokinase in Streptomyces leuoris (a) and formation of levorin in culture medium (b). 0 Indicates use of a medium with 0.4 mM KH2P04;0 indicates a medium with 4.0 mM KH2P04.

in the cells. For bacteria, this was first demonstrated by Harold (1966), then in our laboratory (Nesmeyanova et al., 1973a,b; 1974a,b; 1975b;Maraeva et af., 1979) as well as by other researchers (Yagil, 1975; Zuckier et al., 1980; Tommassen and Lugtenberg, 1980; Argast and BOOS,1980). Investigations conducted with E. cofi showed that, when these bacteria are placed in a fresh medium without orthophosphate, the level of polyphosphates in cells drops drastically (Fig. 15), and the subsequent addition of orthophosphate to the culture starved of phosphorus restores the initial polyphosphate level. The involvement of polyphosphates in regulation of the intracellular orthophosphate concentration in E. cofi is also supported by the fact that synthesis of polyphosphatase participating in polyphosphate hydrolysis is induced during phosphorus starvation simultaneouslywith other phosphohydrolases including tripolyphosphatase and alkaline phosphatase (Nesmeyanova et al., 1974a) as well as acid phosphatase (Maraeva et al., 1978).

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

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P v

u-

0

In

L

C 0

E

.m

c

C 0

W c 0

c 111 n c

a 60

I80 Time (min)

300 Time (min)

FIG. 15. Effect of exogenous orthophosphate on the concentration of intracellular orthophosphate and polyphosphate in Escherichiu colt (a) describes behaviour in a medium with excess of orthophosphate;and (b) with a deficiency of orthophosphate. 1 describes culture growth, 2 polyphosphate concentration (pg (mg dry weight)-’), 3 intracellular orthophosphate concentration (pg (mg dry weight)-’) and 4 the concentration of orthophosphate in the medium.

An identical response of different phosphohydrolases to concentrations of exogenous orthophosphate points to a common character in their functions connected, apparently, with regulation of orthophosphate level in the cells of this bacterium. The unity of function of different phosphohydrolases manifested in the orthophosphate requirements of E. coli was also confirmed by genetic studies. Using E. coli mutants for regulatory genes for alkaline phosphatase, Nesmeyanova et ul. (1975b, 1978) and Maraeva et ul. (1978) showed that polyphosphate phosphohydrolases are controlled by the same regulatory genes as alkaline phosphatase, thus forming a common phosphate regulon together with a number of proteins involved in phosphate metabolism. Such proteins include a phosphate-binding protein (Willsky and Malamy, 1976), one binding glycerophosphate (Argast and Boos, 1980), and one of the proteins of the outer membrane of E. coli, namely protein “e” which is supposedly involved in phosphorylation of pores specific for orthophosphate and its polymers (Argast and Boos, 1980; Tommassen and Lugtenberg, 1980). Comparison of these results points convincingly to the fact that in

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

living organisms, even such primitive ones as bacteria, regulation of intracellular orthophosphate concentration is subtle even at the genetic level. Control of regulation of orthophosphate concentrations is also explained by the fact that the same cells can contain several “metabolic traps” transforming excess intracellular orthophosphate into a polymerized form. To cite one example, in the fungus P . chrysogenum (Okorokov and Kulaev, 1968) and in other organisms (Okorokov et al., 1970, 1973a,b), polymeric complex compounds of phosphorus with various divalent metal cations (Fez+,Mg2+,Caz+,Co2+and others) were detected together with condensed polyphosphates in which, as we have already seen, orthophosphate residues are linked by the energy-rich phosphoanydride bonds. Investigations of the properties of the complexes isolated suggested that their phosphate residues are linked not by covalent but by co-ordination bonds via metal ions. Many organisms proved to have notable amounts of such polymeric metal phosphates. They occur in cells frequently together with high molecular-weight polyphosphates (Okorokov et ul., 1973b). Hence, orthophosphate released by various biochemical reactions may be bound either through formation of polymeric metal phosphate complexes or with the help of reaciions leading to condensed phosphates (polyphosphates). However, it is important to note that the two pathways of orthophosphate polymerization in the cell differ markedly in their energy requirements, i.e. in contrast to formation of polymeric metal phosphate complexes, polyphosphate biosynthesis requires an additional energy supply to form the macroergic phosphoanhydride bonds. Therefore, under some conditions, regulation of free phosphate concentrations may proceed primarily at the expense of polyphosphate formation in cells. In other cells, polymeric metal phosphates may accumulate, especially those that are, at that time, in excess in the cells and their environment. All of the above considerations refer in the first place to eukaryotes and above all to fungi in which, in addition to polyphosphatases, metal phosphate complexes have been detected. In prokaryotes, in particular in blue-green algae, it is high molecular-weight polyphosphates that are mainly involved in regulating intracellular concentrations of both phosphate anions and many cell-absorbed cations. This conclusion is supported by the investigations already referred to and conducted in Jensen’s laboratory (Sicko-Goad et al., 1975, 1978; Baxter and Jensen, 1980a,b). Improved techniques of X-ray dispersion analysis combined with electron microscopy provided the most detailed information on this problem (Baxter and Jensen, 1980b). This work revealed the ability of the cyanobacteria studied to take up and concentrate in polyphosphate granules divalent metals, such as magnesium, barium and manganese. It is interesting that strontium is accumulated in cells of P . boryanurn not in polyphosphate bodies but in some other electron-dense

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granules containing, instead of phosphorus, sulphur as well as potassium and some calcium. In other experiments (Sicko-Goad et al., 1975, 1978), it was found that calcium ions are also often present in polyphosphate granules. The cation composition of polyphosphate granules in cyanobacteria was found to vary markedly depending on the content of specific cations in the environment. These data suggest that, in prokaryotes, polyphosphates play a very important part in regulating the concentration in cytosol not only of phosphate but also of various metals. It is interesting that in some bacteria, such as Desulfooibrio gigas, low molecular-weight polyphosphates appear to be important in binding excess cations. In particular, Jones and Chambers (1975) isolated from D. gigas granules contaning pure magnesium tripolyphosphate. The physiological significance of the accumulation in prokaryotic cells (as well as in those of eukaryotes) of a large amount of phosphate and, respectively, of specific cations in the form of granules secluded from cytosol, consists in the maintenance of stable, usually rather low, intracellular concentrations of monomeric phosphate and free cations. The question arises concerning the purpose of such a maintenance. In fact, excess accumulation in cells of low molecular-weight compounds, e.g. glucose, amino acids, phosphate, or some cations, may drastically affect the intracellular osmotic pressure and pH value. At the same time, such compounds as AMP, ADP, ATP, acetyl-CoA, Mg2+, NADP, phosphate and glucose are, in certain concentrations, potent effectors and regulators of the functioning of important enzymic systems in the cell. In this connection, in the course of evolution organisms have developed systems of neutralizing excess amounts of physiologically active monomers, i.e. specific “metabolic traps”. In our opinion, such traps function by the processes involved in polymerization of corresponding monomers to glycogen, polyphosphates, poly-8-oxybutyric acid, cyanophycin or polymeric metal phosphates. It seems probable that similar processes involved in detoxication of osmotically active compounds (acetyl-CoA, organic and amino acids) are pathways leading to secondary metabolites including polyphenols, isoprenoids, antibiotics and alkaloids. In concluding the discussion of polyphosphate metabolism in prokaryotes, we will dwell on two other aspects. Recently, data were published on bacteria populating specific ecosystems. Firstly, Bobyk et al. (1980) investigated some particular features of polyphosphate metabolism in Bdellouibrio bacteriouorus, a parasite living on E. coli and some other bacteria. It was found that, in these parastic bacteria, the amounts of polyphosphates are several times higher than that in the host cells, and B. bacteriouorus contained predominantly the acid-insoluble, i.e. surface-localized, fraction of polyphosphates. As already mentioned, certain enzymes of polyphosphate metabolism were detected in these parasites. The activity of 1,Iphosphoglycerate: polyphos-

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phate phosphotransferase and polyphosphatase was much higher than in cells of E. coli, in the periplasm of which they generally live. The fact that the amount of polyphosphates and the intensity of their metabolism in the parasite appears to be higher than in host cells with the B. bacferiovorus-E. coli system brings to mind a similar situation observed by Bennett and Scott (1971) during studies of the wheat stem rust fungus infecting wheat leaves. Secondly, Egorova et al. (198 1) demonstrated substantial amounts of polyphosphates and ATP in the extremely thermophilic bacterium Thermus Jlauus 71, normally growing at 65-70°C. Their concentrations in Thermus flaws 71 exceeded several times those in E. colicells. It is interesting that, in T. Jlavus, polyphosphates are represented mainly by low molecular-weight fractions (PolyP~and PolyPz), i.e. fractions usually localized inside the cytoplasmic membrane. Thirdly, Nikitin et al. (1979, 1983) reported interesting results on analyses of polyphosphate and ATP contents in the oligotrophic bacteria Renobacter vacuolatum and Tuberoidobacter mutans which populate the atmosphere and exhibit very slow growth with the scanty nutrients available in the air. Little ATP and tremendous amounts of polyphosphates, mainly acid-insoluble, were detected in both cases. Also, in R . vacuolatum, 0.54.7 pmol of ATP (g dry wt)-’ and 220-280 pmol of polyphosphates were detected, i.e. the ATP concentration was one order of magnitude lower compared with common eubacteria (e.g. E. coli), whereas that of polyphosphates was one order of magnitude higher compared with concentrations usually detected in bacteria. These findings, as well as other information available in the literature (see, e.g., Sudyina et al., 1978; Kulaev, 1979),enable one to infer that the amounts of polyphosphate fractions and their importance in metabolism vary in bacteria and other organisms and depend greatly on ecological factors. Another aspect of polyphosphate metabolism, which should be given at least brief consideration, is the intensive accumulation of high molecularweight polyphosphates in Acinetobacter sp. isolated from sedimentation tanks containing waste waters of certain industries (Fuhs and Chen, 1975). These bacteria are able to take up from the medium tremendous amounts of phosphate without prior starvation of phosphorus. They can absorb phosphate from sewage waters containing large concentrations of phosphate and accumulate it in the form of polyphosphates. This phenomenon was called “luxury uptake” (Levin and Shapiro, 1965). Deinema et al. (1980) found that the bacteria which absorb phosphate from phosphate-rich media containing butyrate or acetate as a carbon source accumulate large amounts of highly polymerized polyphosphates and lipids. After 40 hours’ growth, the phosphate content was 10-20% and the lipid content was up to 25% of dry weight, and bacterial cells were literally stuffed with polyphosphate and lipid granules. Investigation of these bacteria is of exceptional interest in view of their

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possible use in extraction of phosphate from waste waters discharged into inland water bodies in industrialized countries. E. CONCLUDING REMARKS O N THE PHYSIOLOGICAL ROLE OF HIGH MOLECULAR-WEIGHT POLYPHOSPHATES IN MICROBIAL METABOLISM

Summing up, in spite of all that has been stated about the possible physiological role of high molecular-weight polyphosphates in the activities of organisms, it should be underlined that they are regulators of the intracellular concentration of important metabolites including ATP, ADP, other nucleoside polyphosphates, and finally pyro- and particularly orthophosphate. Moreover, they represent a valuable pool of activated phosphate, which can be utilized in various metabolic processes, primarily in those connected with different stages of carbohydrate and nucleic acid metabolism; transport and oxidation of carbohydrates, biosynthesis of cell-wall polysaccharides, and biosynthesis, degradation and functioning of nucleic acids. It is in micro-organisms that high molecular-weight polyphosphatesplay an exceptional role. This is basically explained by two circumstances. First, unlike higher organisms, they do not have a well-developed system of hormonal and nervous regulation; second, micro-organisms depend very much on environmental conditions, resulting from direct contact of cells with the surrounding medium. An impoverished set of regulatory mechanisms in micro-organisms must obviously lead, under certain conditions, to insufficiently finely balanced biochemical reactions. Therefore, micro-organisms should have “metabolic traps” such as high molecular-weightpolyphosphates capable of maintaining their intracellular homoeostasis. The need for “metabolic traps” is also due to a very strong dependence of micro-organisms on environmental conditions. When growth and development of microorganisms depend directly on the environment, it appears very important for the organism to be able to enhance its vital activities immediately on creation of favourable conditions. The availability of sufficient amounts of such valuable endogenous pools as high molecular-weight polyphosphates makes micro-organisms,on the one hand, less dependent on external conditions and, on the other hand, capable, at any suitable moment, of initiating growth and reproduction without any considerable lag-period. In higher organisms, the role of such phosphorus compounds in metabolism is apparently less essential. This inference may be supported by the poor accumulation of polyphosphates in tissues of higher plants and animals and the availability of only a limited number of enzymes for polyphosphate metabolism. It may be assumed that, in highly organized organisms, polyphosphates perform some quite specialized functions, being donors of

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activated phosphate only for quite specific biochemical or physiological processes. Such is, in general words, today's position regarding the physiological role of high molecular-weight polyphosphates in the vital activities of contemporary organisms.

111. Inorganic Pyrophosphate: New Aspects of its Metabolic and

Physiological Role

Inorganic pyrophosphate is orthophosphate anhydride, which can be considered the least polymeric polyphosphate with an ii value of 2. The free energy of pyrophosphate hydrolysis is close to that released as a result of splitting terminal phosphate groups from ATP and ADP. However, in the presence of bivalent cations, the value of the free energy of pyrophosphate hydrolysis (AGO') is somewhat lower than that for ATP and ADP. According to Lawson et al. (1976), in the presence of 1 mM Mg2+,pH 7.0 and 38"C,the AGO' value of ATP hydrolysis to ADP is 31.8 kJ mol-I (7.6 kcal mol-I) and that of pyrophosphate hydrolysis is 22.1 kJ mol-I (5.27 kcal mol-I). Taking into account the cation concentration in Entamoeba histolytica, Reeves et al. (1974) obtained a AGO' value of 25.1 kJ mol-I (6.0 kcal mol-') for pyrophosphate hydrolysis. Under similar conditions, Flodgaard and Fleron (1 974), in experiments on liver cells, obtained lower values for pyrophosphate, i.e. about 16.7 kJ mol-I (4 kcal mol-I). Thus, from the thermodynamic point of view, pyrophosphate can be a potential source of energy in phosphorylation reactions. It was, however, believed for a long time that pyrophosphate is only a byproduct of numerous reactions of pyrophosphorolysis involved in biosynthesis of proteins, nucleic acids, lipids, polysaccharides and nuceloside coenzymes. It was thought that, due to the activity of cellular pyrophosphatases, pyrophosphate could not be accumulated in the cell and was hydrolysed to orthophosphate, thus ensuring that the above reactions were irreversible (Kornberg, 1957, 1959; Hoffmann-Ostenhof and Slechta, 1957). A. UTILIZATION OF PYROPHOSPHATE IN PHOSPHORYLATION REACTIONS I N BACTERIA

The first reaction in micro-organisms in which pyrophosphate was shown to be utilized as a source of phosphorylation, thus replacing ATP, was that revealed by Siu and Wood (1962) in Propionibacterium shermanii. This

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reaction is catalysed by the pyrophosphate (PPi)-dependent phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.38):

+

PPi oxaloacetate+PEP +Pi +COz

(1)

Pyrophosphate-dependent phosphoenolpyruvate carboxykinase reaction (1) is similar to the following reaction: ATP + oxa1oacetateePEP + ADP + CO2

(2)

which is catalysed by phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) earlier discovered by Utter and his coworkers (Utter and Kurahashi, 1954; Utter et al., 1954) and widely found in nature (Scrutton and Young, 1972). As shown by Wood et al. (1966), pyrophosphate-dependent carboxykinase (EC 4.1.1.38) operates in P . shermanii as phosphoenolpyruvate carboxykinase (EC 4.1.1.32), especially in bacteria grown on lactate. In the protozoan E. histolytica, the polyphosphate-dependent enzyme could also substitute for the lack of phosphoenolpyruvate carboxykinase (GTP) (Reeves, 1970, 1976). Later, this enzyme was found in Rhodopseudomonas palustris (Chernyadyev et al., 1972) and Brevibacterium ammoniagenes (Baryshnikova and Loginova, 1979). In Rh. rubrum, pyrophosphate-dependent carboxykinases are active only when bacteria are grown in the light in the presence of malate, i.e. under conditions of active pyrophosphate synthesis by Rh. rubrum cells (Shady et al., 1975). Another reaction occurs in micro-organisms in which pyrophosphate is a phosphorylating agent. This reaction is catalysed by pyruvate, phosphate dikinase (EC 2.7.9.1) and proceeds as follows:

+

+

PPi AMP PEP+pyruvate+ ATP +Pi

(3) This enzymic pathway of pyrophosphate utilization has been found in propionic bacteria (Evans and Wood, 1968), E. histolytica (Reeves, 1968)and Bacteroides symbiosus (Reeves et al., 1968; Reeves, 1971). Later, this enzyme was found in Acetobacter suboxydans cultured on substrates of the tricarboxylic acid (TCA) cycle (Benziman and Palji, 1970; Benziman and Eisen, 1971) and in some photosynthetic bacteria (Buchanan, 1974). The pyruvate, phosphate dikinase reaction (3) is similar to the reaction catalysed by pyruvate kinase (EC 2.7.1.40): ADP +PEPepyruvate + ATP

(4)

In B. symbiosus and E. histolytica, pyruvate phosphate dikinase is involved in glycolysis (Reeves, 1968, 1976; Reeves et al., 1968; Wood, 1977; Wood et al., 1977) substituting for pyruvate kinase which these organisms lack. In this situation, pyrophosphate acts as a direct source of high-energy phosphate required for ATP biosynthesis. The functioning of these two enzymes depends

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directly on the conditions under which micro-organisms are grown. In A. suboxydans, pyruvate phosphate dikinase can be found only when organisms are cultured in a medium containing pyruvate or TCA cycle substrates. A study of the regulation of pyruvate kinase and pyruvate phosphate dikinase in A. suboxydans showed that activities of the two enzymes are regulated in an opposite manner, depending on the energy state of the cell, particularly on the ratio of concentrations of adenine nucleotides, i.e. AMP, ADP and ATP. Pyrophosphate-dependent phosphofructokinase (EC 2.7.1.9.0), which is responsible for the reversible reaction:

+

Fructose 6-phosphate + PPiefructose 1,6-diphosphate Pi

(5)

was detected and studied in E. histolytica (Reeveset al., 1976)and P . shermanii (O’Brien et al., 1975). Recently this enzyme was found in marine organisms Alcaligenes sp. and Pseudomonas marina (Sawyer et al., 1977)and Bacteroides fragilis (Macy et al., 1978). The above reaction ( 5 ) is similar to that catalysed by ATP-dependent phosphofructokinase:

+

ATP + fructose 6-phosphateefructose 1,6-diphosphate ADP

(6)

It is interesting to note that activities of pyrophosphate-dependent phosphofructokinase in P . shermanii and E. histolytica are one order of magnitude higher than those of the ATP-dependent enzyme. Apparently, in propionic bacteria grown on glucose, pyrophosphate essentially replaces ATP in synthesis of fructose 1,ddiphosphate (O’Brien et al., 1975). The transformation of fructose 1,Qdiphosphate takes place mostly due to activity of phosphofructokinase to form pyrophosphate (by the reverse of reaction 5) compared to hydrolysis by fructose diphosphatase, as the activity of the latter is 1 5 2 0 times lower than that of pyrophosphate-dependent phosphofructokinase. Attempts to detect pyrophosphate-dependent phosphofructokinase in yeast failed. Konoshenko et al. (1979) demonstrated that pyrophosphate is a competitive inhibitor of ATP-dependent phosphofructokinase. In addition to the above enzymes, in P . shermanii, P . technicum and P. freudenreichii, an enzyme responsible for direct phosphorylation of serine to 0-phospho-L-serine (EC 2.7.1.80) (Cagen and Friedmann, 1968, 1972) has been detected:

+

PPi serine*phosphoserine

+Pi

(7)

A pyrophosphate-dependent acetyl kinase was found in E. histolytica to catalyse a reaction similar to the ATP-dependent acetyl kinase reaction (Reeves and Guthrie, 1975):

PPi + acetate$acetyl phosphate + Pi

+

ATP acetategacetyl phosphate +Pi

(8) (9)

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145

It was then reported that two microsomal polypeptides can be phosphorylated at the expense of pyrophosphate (Lam and Kasper, 1980a,b). All of these data point to an important role being played by pyrophosphate as a compound which, in certain cases, can successfully replace ATP in phosphotransferase processes in micro-organisms. At the same time, a pyrophosphate-dependent glucokinase, which catalyses phosphorylation of glucose to glucose 6-phosphate with pyrophosphate, has not hitherto been detected in micro-organisms (Nordlie and Arion, 1964; Stetten, 1964; Stetten and Tafft, 1964; Nordlie, 1976). More detailed information on the enzyme involved in phosphorylation reactions can be found in the reviews by Reeves (I 976), Wood (1977), Wood et al. (1977) and Mansurova (1982), as well as in other recent publications (Milner et al., 1978; Moscovitz and Wood, 1978; Yoshida and Wood, 1978). B . ENERGY-DEPENDENT SYNTHESIS OF PYROPHOSPHATE DURING PHOTOSYNTHETIC A N D OXIDATIVE PHOSPHORYLATION

The first evidence for an energy-dependentbiosynthesis of pyrophosphate was obtained with animal tissue homogenates (Cori, 1942; Cross et al., 1949). At the end of the 1950s, Klungsoyr and his colleagues (Klungsoyr et al., 1957; Klungsoyr, 1959) demonstrated intensive incorporation of [32P]orthophosphate into pyrophosphate by aerated cells of A. suboxydans, E. coli and Merrulins lacrimans. At the same time, Shaposhnikov and Fyodorov (1960), working on the green sulphur bacterium Chlorobium thiosulphatophilum, showed that under illumination in the absence of carbon dioxide [32P]orthophosphate was incorporated into polyphosphates of the acid-soluble fraction at a high rate. However, the authors did not identify the particular compound. In 1966, Baltscheffsky and his colleagues (Horio et al., 1966; H. Baltscheffsky et al., 1966; M. Baltscheffsky et al., 1966; Baltscheffsky and Stedingk, 1966) reported that chromatophores of the non-sulphur purple bacterium Rh. rubrum could synthesize pyrophosphate as an alternative to ATP and utilize it in reactions occurring at the level of the photosynthetic electron-transport chain. Almost simultaneously, light-dependent synthesis of pyrophosphate was found in cells of Chlorella sp. and spinach chloroplasts (Pedersen et al., 1966a,b). It should be noted that Rh. rubrum cells, grown in the light, contained pyrophosphate either in the same or far greater amounts than ATP. Pyrophosphate was not detected in an acid extract from the same cells grown in the dark (Shady et al., 1975). The experimental data available suggest that pyrophosphate-dependent energy metabolism is inherent not only in Rh. rubrum but also in Rh. palustris and Rh. viridis (Chernyadyev et al., 1972; Knobloch, 1975; Jones and

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IGOR S. KULAEV AND VLADlMlR M. VAGABOV

Sanders, 1972) and probably in other photosynthetic micro-organisms (Shaposhnikov and Fyodorov, 1960). An outstanding contribution to a proper understanding of the important role that pyrophosphate may play in the bioenergetic processes of the cell was made by H. Baltscheffsky and his coworkers. They demonstrated that, in the absence of ADP, chromatophores of Rh. rubrum carry out light-dependent synthesisof pyrophosphate (Horio et al., 1966; H. Baltscheffsky et al., 1966; M. Baltscheffsky et al., 1966). It was shown that syntheses of both pyrophosphate and ATP depend on photosynthetic electron transport and are inhibited by antimycin and uncouplers. However, there is an important difference between synthesis of pyrophosphate and of ATP. Oligomycin does not inhibit synthesis of pyrophosphate and sometimes stimulates it slightly; moreover dicyclohexylcarbodiimide had no effect on synthesis of pyrophosphate, whereas Di 0-9 inhibits that of both compounds (Guillory and Fisher, 1972). The Baltscheffskys (H. Baltscheffsky et al., 1966, 1969, 1971; M. Baltscheffsky, 1969a,b; H. Baltscheffsky and M. Baltscheffsky, 1972;Baltscheffsky, 1977), Keister and his colleagues (Keister and Yike, 1967a,b; Keister and Minton, 1971a,b; Rao and Keister, 1978) and Skulachev and his colleagues (Isaevet al.?1970,1976;Kondrashinet al., 1980;Skulachev l971,1972a, 1975; Ostroumov et al., 1973) have described in detail the mechanism of pyrophosphate biosynthesis and utilization at the level of the electron-transport chain. The reaction sequence is presented in Fig. 16. Cyclic transport of electrons results in formation of a non-phosphorylated high-energy intermediate or a certain energized state of the membrane whose energy can be used to maintain ion transport, reverse electron transfer and transhydrogenase reaction of pyrophosphate-dependent NAD+ reduction, to modify the conformation of carotenoid molecules leading to changes in their absorption spectrum, and, finally, to synthesize ATP and pyrophosphate (H. Baltscheffsky et al., 1966; M. Baltscheffsky et al., 1966; M. Baltscheffsky, 1969a,b, 1971, 1974, 1977; Keister and Yike, 1967a,b; Keister and Minton, 1971a,b;Azzi et al., 1971; Fischer and Guillory, 1969a,b). A detailed survey of the problem can be found in a recent publication by Baltscheffsky (1978). Baltscheffsky and Stedingk (1966) hypothesized that the resultant pyrophosphate can be further used in biosynthesis of inorganic polyphosphates. The most important inference from this work is that pyrophosphate is a product of photophosphorylation in chromatophores as an alternative to ATP, and that membrane pyrophosphatase is a factor coupling electron transport and pyrophosphate synthesis. These conclusions are supported by studies in Saccharomyces cerevisiae and a yeast-like fungus Endomyces magnusii (Mansurova et al., 1975b, 1977a, 1978).It appears that mitochondria of these organisms synthesize, during oxidative phosphorylation, not only ATP but pyrophosphate as well. Inhibitors of the respiratory chain, such as antimycin

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

147

Light

Bacteriochlorophy ll Cyclic electron- transport chain

-X ,

quinone

cytochrome b

Change in the spectrum of carotenoids transhydrogenase reaction

PH

PPI

ATP

FIG. 16. Mechanism of light-dependent biosynthesis of polyphosphate in chromatophores of Rhodospirillum rubrum and its utilization in dark reactions. From Mansurova (1982).

and cyanide, and the uncoupler 2,4-dinitrophenol inhibit both synthesis of ATP and pyrophosphate. Oligomycin inhibits only ATP synthesis, whereas sodium fluoride inhibits only pyrophosphate synthesis. It can be concluded that, in mitochondria, pyrophosphate synthesis depends on the respiratory chain and that ATP does not act as pyrophosphate precursor. It appears that maximal synthesis of ATP and pyrophosphate in mitochondria and chromatophores requires different conditions. For instance, this operates with respect to the rate of electron flow along the photosynthetic and respiratory electron-transport chains, and to the redox potential of electron carriers (Horio et al., 1965; Horiuti et al., 1968; Nishikawa et al., 1973; Pullaiach et al., 1980). It can be postulated (Pullaiach et al., 1980) that phosphorylation yielding pyrophosphate takes place when ATP synthesis becomes restricted or impossible. As already mentioned, membrane-bound pyrophosphatase participates in energy-dependent synthesis of pyrophosphate. Rhodospirillum rubrum contains at least two pyrophosphatases; one cytoplasmic (Klemme and Gest, 1971a,b) and the other membrane-bound (M. Baltscheffsky et al., 1966;

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IGOR S. KULAEV A N D VLADlMlR

M . VAGABOV

Fischer and Guillory, 1969a; Isaev et al., 1970; Keister and Minton, 1971a,b; Guillory and Fischer, 1972; Dutton and Baltscheffsky, 1972). The soluble enzyme has entirely different properties from the membrane one. The two pyrophosphatases were found in mitochondria of all organisms studied; including yeast, fungi and animal tissues (Irie et al., 1970; Kulaev et al., 1973c; Umnov et al., 1974b). It should be noted that the chromatophore pyrophosphatase activity can be manifested only in a lipid environment. Detergent extraction of the enzyme from membranes followed by removal of phospholipid results in complete inactivation. However, the enzyme can be reactivated in the presence of phospholipids (Isaev et al., 1970; Kondrashin et al., 1980). Kondrashin et al. (1980) showed that membrane pyrophosphatase isolated from chromatophores can be incorporated into liposomes and generate a membrane electrochemical potential on addition of pyrophosphate. It is so far unclear what is the nature of the common intermediate for ATP and pyrophosphate synthesis. Recent data suggest that the common intermediate in the synthesis of ATP and pyrophosphate is, in agreement with Mitchell’s views (Mitchell, 1961, 1966, 1968), an energized state of the membrane with an electrochemical potential across it. Inorganic pyrophosphate is one of the components of the common energy pool of the cell, and the energy of the phosphoanhydride bond can be transferred from ATP to polyphosphate and back through intermediate formation of the electrochemical potential. The validity of reaction sequences leading to ATP and pyrophosphate (Fig. 17) has been convincingly proved by the studies of pyrophosphate-dependent

Adenosine triphosphatase

AT P

A~u.H+

Pyrophosphatase

PP

FIG. 17. Mechanism for the energy-dependent synthesis of ATP and pyrophosphate (PP) in chromatophores, chloroplasts and mitochondria. A and B are components of the electron transport chain.

POLYPHOSPHATE METABOLISM IN MICRO-ORGANISMS

149

synthesis of ATP and ATP-dependent synthesis of pyrophosphate carried out by Keister and his colleagues (Keister and Minton, 1971a,b; Keister and Raveed, 1974;Baltscheffsky and Baltscheffsky 1972; Mansurova et al., 1973a, 1975a,c). Of great importance is the fact that these reactions took place with simultaneous involvement of ATPase and pyrophosphatase and when the respiratory chain was switched off. In this situation, only the energy of the phosphoanydride bond of ATP or pyrophosphate is used, and the orthophosphate residue is not transferred. For instance: 1. PPi

pyrophosphau

2. ADP +"Pi

+

,2pi + "

"N "

N

"

ATPase

-ATP3'

The data obtained indicate that, in chromatophores and mitochondria, the content of ATP and pyrophosphate are in equilibrium, established with participation of coupling ATPase and pyrophosphatase. In chromatophores, synthesis of one molecule of ATP is accompanied by hydrolysis of 10 molecules of pyrophosphate (Keister and Minton, 1971a,b). It should be mentioned, however, that, in addition to the coupling membrane pyrophosphatase, chromatophores contain an active cytoplasmic membrane which also contributes to pyrophosphate hydrolysis. The intensity of ATP and pyrophosphate production is, to a great extent, affected by the physicochemical state of the membrane. It was shown that the rate of ATP and pyrophosphate synthesis depends directly on the viscosity of the phospholipid component of the mitochondria1inner membrane (Kulaev et al., 1980; Mansurova ef al., 1982). Synthesis of ATP increases with decreasing membrane fluidity, and inversely, increasing membrane fluidity favours pyrophosphate synthesis. The literature indicates that light-dependent synthesis of pyrophosphate can be performed not only by photosynthetic bacteria but also by chloroplasts of algae and higher plants (Rubtsov et al., 1976). Both in chromatophores (Guillory and Fischer, 1972) and in chloroplasts (S.E. Mansurova, personal communication), maximal synthesis of pyrophosphate occurs at a lower illumination than that required for ATP. Thus, data obtained in recent years on pyrophosphate metabolism in micro-organisms and the occurrence of energy-dependent synthesis of pyrophosphate in both mitochondria of lower organisms (yeasts) and higher eukaryotes (mammals) (Mansurova et al., 1973a,b, 1975a,b, 1976, 1977a,b), as well as in chloroplasts of algae and higher plants (Rubtsov et al., 1976) enable one to regard pyrophosphate not only as a byproduct of pyrophosphorolysis reactions used in the bioenergetics of the most ancient forms of life but also as a high-energy compound similar to ATP, involved in storage and

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IGOR S. KULAEV AND VLADlMlR M . VAGABOV

utilization of energy in contemporary highly organized micro-organisms. Investigating the role of pyrophosphate in metabolism has turned from a matter of elucidating the peculiarities of the energetics of the most ancient life forms and revealing the manifestations of “predeluvian metabolism” (Wood, 1977; Wood et al., 1977; H. Baltscheffsky, 1971) into a problem of general biological interest. C . RELATIONSHIP BETWEEN PYROPHOSPHATE A N D POLYPHOSPHATE

METABOLISM IN MICRO-ORGANISMS

The Italian authors Ipata and Felicioli (1963) were the first to raise this question. They reported enzymic phosphorolysis of high molecular-weight polyphosphates to pyrophosphate in yeast. We attempted to reproduce these experiments and failed to demonstrate an enzymic character of the reaction (Mansurova et al., 1973; Kulaev and Skryabin, 1974):

+

(Polyp),+ 32Pi+32PPi (Polyp),-

1

We showed that, in the presence of divalent cations, significant quantities of radioactive pyrophosphate were formed nonenzymicallythrough phosphorylation of [32P]orthophosphateby high molecular-weight polyphosphate. Pyrophosphate can be synthesized enzymically from tripolyphosphate by means of a specific enzyme, tripolyphosphatase. This enzyme has been detected in many organisms, including Aspergillus oryzae (Neuberg er ul., 1950), Neurospora crassa (Kulaev and Konoshenko, 1971b), Phyrophrhoru infestans (Sysuev et al., 1978), yeast (Mattenheimer, 1956a,b,c; Felter and Stahl, 1970), Aerobacter aerogenes (Dawes and Senior, 1973), Bacillus sp. (Szymona and Zajac, 1969) and E. coli (Nesmeyanova et al., 1973a).A study of the intracellular localization of the enzyme in yeast and N . crussu demonstrated its presence in vacuoles (Schwencke, 1978), periplasmic space (Kulaev et al., 1972b; Konoshenko et al., 1973) and, to a large extent, in mitochondria (Kulaev et al., 1972b; Konoshenko et al., 1973; Umnov ef ul., 1974b). The enzyme from N . crussa was purified to homogeneity. Egorov and Kulaev (1976) convincingly demonstrated that tripolyphosphate hydrolysis by tripolyphosphatase takes place according to the equation: PPPi +PPi

+Pi

In recent years, relationships between the metabolism of pyrophosphate and that of acid-soluble polyphosphates were given special study (Mansurova, 1979; Ermakova et al., 1981). It was found that, during yeast growth, the pyrophosphate content varies drastically forming two maxima of accumulation at the beginning and at the end of the exponential phase of growth

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(Ermakova er al., 1981; Shakhov et al., 1978). This pattern was also seen during growth of the yeast hybrid strain Sacch. cerevisiae N.C.Y.C. 644 SU3 with and without aeration in media containing various concentrations of glucose, and during aerobic cultivation of Candida guilliermondii on glucose or petroleum hydrocarbon-containing media (Shakhov et al., 1978). Peak pyrophosphate accumulation was not related to changes in the rates of respiration or fermentation. The pyrophosphate content of the yeast Sacch. cerevisiue exceeded the ATP content at different stages by a factor of 10-1000, reaching 2-17 mg (g dry wt)-'. It can be seen that accumulation of such large amounts of pyrophosphate is not associated with bioenergetic processes, and it can be assumed that its major function, as with certain fractions of highly polymeric polyphosphates, is as a form of energy and phosphorus reserve in the cell. When the content of pyrophosphate reached a maximum, that of acid-soluble polyphosphates with higher molecular weights dropped to a minimum (Fig. 18). Microscopic examination of the yeast showed that marked variations in the contents of pyrophosphate and other acid-soluble polyphosphates during growth are associated with significant synchronization of cell budding (Ermakova et al., 1981). Pyrophosphate accumulation is particularly active in cells having a large number of small intensively growing buds. These data are in good agreement with the findings of Nurse and Wiemken (1974) who observed accumulation of low molecular-weight

2oFt tI t

--1 25

Growth (hours)

FIG. 18. Changes in the content of pyrophosphate ( 0 )and acid-soluble polyphosphates without pyrophosphate (0) during growth in aerated (a) and non-aerated (b) cultures of Succh. cerevisiue N.C.Y.C. 644 SU3. From Ennakova et al. (1981).

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AND VLADlMlR M. VAGABOV

substances at the beginning of bud formation in the yeast. As the buds approached the size of the mother cell, the content of pyrophosphate decreased dramatically and that of acid-soluble polyphosphates increased. These polyphosphates are located within the cell, primarily in vacuoles (Indge, 1968; Urech et af., 1978; Schwencke, 1978; Cramer et al., 1980) and, in contrast to other polyphosphate fractions, their behaviour is opposite to that of pyrophosphate throughout the entire period of cultivation. It is unclear so far how acid-soluble polyphosphates are utilized. It is probable that their high-energy phosphate groups can be used in synthesis of ATP and other nucleoside triphosphates by means of polyphosphate: ADP phosphotransferase (EC 2.7.4.1) or be transformed into inorganic pyrophosphate through direct degradation by polyphosphate depolymerase (EC 3.6.1.10) and tripolyphosphatase. It should be noted that three enzyme activities are present in yeast vacuoles (Shabalin et af.,1977; Schwencke, 1978). It is likely that the resultant pyrophosphate is used as an energy source during cation transport through the tonoplast (Okorokov et af., 1980). In addition, degradation products of vacuolar polyphosphates, in particular tripolyphosphate, pyrophosphate and orthophosphate, may participate in transport of arginine and other positively charged molecules across the membrane, as conjectured for yeast (Diirr et af., 1979; Matile, 1978; Okorokov et af., 1980). Having discovered light-dependent synthesis of pyrophosphate in Rh. rubrum chromatophores, Baltscheffsky and Stedingk (1966) assumed that it may take part in the biosynthesis of high molecular-weight polyphosphates. However, our study of polyphosphate and pyrophosphate metabolism in this organism (Kulaev et al., 1974c; Shady et af., 1976) failed to support this concept. In fact, culturing Rh. rubrum in light led to accumulation of significant quantities of pyrophosphate and salt-soluble polyphosphates. Synthesisof high molecular-weight polyphosphates depended on the electrontransport chain and was inhibited by antimycin (Table 11). TABLE 11. Synthesis of ATP, pyrophosphate and salt-soluble polyphosphates by chromatophores of Rhodospirihm rubrum in the light Rate of synthesis (32Pcounts min-l) With ADP Without

Without ADP

Compound

inhibitors

antimycin

oligomycin

With

Without inhibitors

antimycin

ATP Pyrophosphate Polyphosphate

99,490 570 19,110

11,570 160 1,370

27,460 570 3,690

200 2700 330

200 1360 000

With

With

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Oligomycin inhibition of ATP synthesis led to a parallel decline in accumulation of this fraction of polyphosphates (Shady et al., 1976). It can be assumed that synthesis of salt-soluble polyphosphates in these bacteria is realized at the expense of ATP by action of ATP: polyphosphate phosphotransferase (EC 2.7.4. l), an enzyme widely distributed among microorganisms, including photosynthetic bacteria (Kulaev, 1979). In this case, the pyrophosphate-polyphosphate relation can be mediated by the adenine nucleotide system. It is appropriate to mention here the work of Butukhanov et al. (1979) in which direct phosphorylation of AMP and ADP at the expense of pyrophosphate was reported: AMP + PPi +ADP

+Pi ADP + P P +ATP ~ +pi

The enzymes responsible for the two reactions differed from the corresponding polyphosphate-dependent enzymes. It may be postulated therefore that, in certain micro-organisms, the energy of the phosphoanhydride bond and sometimes the phosphoric acid residue can be readily transferred from pyrophosphate to polyphosphate and back via adenine nucleotides. However, as already shown, for example, for synthesis of nucleic acids and for some other biosynthetic processes, a direct transfer of orthophosphate residues from pyrophosphate to polyphosphates is not excluded. This process seems to be closely connected topologically with the functioning of those biosynthetic systems in which pyrophosphate is one of the end products. It may be thought that such conjugated systems are analogous to the recently detected multi-enzyme complex of adenylate translocase and creatine phosphokinase in animal mitochondria (Saks et al., 1977, 1980).

IV. Modern Concepts about the Role of High Molecular-Weight Polyphosphates and Pyrophosphate in Evolution of Phosphorus Metabolism At the present time, most geochemists and biologists hold that the earliest living beings on Earth were anaerobic micro-organisms which obtained energy from hexose fermentation to lactate and ethanol (Oparin, 1957). The further course of evolution is debatable. Some researchers believe the fermenting anaerobes were followed by “anaerobically breathing” organisms that had an incipient membranous electron-transport chain (Sagan, 1967; Margulis, 1970; Hall, 1971). In their opinion, mutations of the gene coding for the cytochrome prosthetic group led to emergence of chloro-

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phyll and anaerobic photosynthetic organisms. In contrast, most authors (Schlegel, 1972; Skulachev, 1972b, 1974; Broda, 1971, 1975; Gusev and Gokhlerner, 1980) share the opinion that the first electron-transport chains and the coupling mechanisms of electron transport and phosphorylation developed after anaerobic micro-organisms acquired photosynthetic abilities. Oxygen accumulation in the Earth’s atmosphere could be accounted for by the appearance of photosynthetic cyanobacteria capable of water photolysis (Oparin, 1957; Broda, 1971; Gusev and Gokhlerner, 1980; Wilson and Lin, 1980). The presence in the atmosphere of sufficient quantities of oxygen was responsible for emergence of aerobic organisms that utilized it as the terminal electron acceptor. It is hard to say whether anaerobic respiration was primary or secondary to photosynthesis. It is essential that, at a certain evolutionary stage, glycolytic phosphorylation occurring in solution was paralleled by membrane bioenergetics. It is possible that, on the early Earth, initial high-energy phosphates were represented by high molecular-weight polyphosphates synthesized at high temperatures during volcanic and other processes (Belozersky, 1959; Kulaev, 1971, 1979) and by inorganic pyrophosphate produced either from orthophosphates (Calvin, 1963, 1971; Miller and Parris, 1964; Beck and Orgel, 1965; Lipmann, 1965)or non-biologicallyfrom polyphosphates in an aqueous medium (Mansurova et al., 1973c; Kulaev and Skryabin, 1974). It can be assumed that, when the Earth was surrounded by a reducing atmosphere with a low concentration of oxygen, both high molecular-weight polyphosphates and pyrophosphate were major components of the energy system in primordial organisms. Calvin (1963) and Lipmann (1965) were the first to suggest the participation of pyrophosphate in accumulation and transfer of energy-rich bonds on the primeval Earth. These authors put forward the idea that the reactions typical of primitive forms of life evolved from prebiological systems, and that living organisms of today still have the ability to utilize pyrophosphate as a high-energy compound. This concept found support in our investigations (Mansurova et al., 1975a,c, 1976, 1977a,b; Rubtsov et al., 1976; Mansurova and Ibragimov, 1979) and in experiments carried out by Wood and his colleagues (Wood, 1977; Wood et al., 1977)and other workers (Baltscheffsky et al., 1966; Batscheffsky and Stedingk, 1966; Nordlie and Arion, 1964; Nordlie, 1976; Reeves, 1976). We have demonstrated that contemporary primitive organisms, including bacteria, actinomycetes and fungi, contain an enzyme catalysing transfer of activated phosphate from 1,3-diphosphoglyceratenot to ADP to form ATP but directly to high molecular-weight polyphosphates (Kulaev et al., 1968; Kulaev and Bobyk, 1971). Active synthesis of pyrophosphate at the expense

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of glycolytic phosphorylation has also been observed in yeast (Mansurova et al., 1976). Extensive studies on the distribution of polyphosphate hexokinase among micro-organisms performed in our laboratory have shown that this enzyme occurs only in phylogenetically very ancient and closely related micro-organisms forming a distinct class of actinomycetes according to Krasilnikov’s (1949) evolutionary systematics. It should be emphasized that, in the oldest representatives of these micro-organisms, e.g. micrococci, tetracocci and propionic-acid bacteria (Kulaev, 1979), activity of polyphosphate hexokinase is several times higher than that of ATP hexokinase, whereas in the newest representatives of this class, i.e. true actinomycetes,the activity of ATP hexokinase significantly exceeds that of polyphosphate hexokinase. These data, as well as the findings of Wood et al. (1977), give evidence that, in the best studied propionic-acid bacteria, glycolytic degradation of glucose takes place with participation of polyphosphates and pyrophosphate rather than the ADP-ATP system. It can be expected that, with increasing importance of membrane-bound energy processes in primitive orgaisms, the bioenergetic role of ATP and pyrophosphate will become more significant while that of polyphosphates will correspondingly decrease. It is far from incidental that high molecular-weightpolyphosphates, and enzymes of their metabolism, are absent from chloroplasts of algae and higher plants (Rubtsov and Kulaev, 1977; Rubtsov et al., 1977) and mitochondria (Kulaev et al., 1967b) which, according to the theory of symbiogenesis of eukaryotic cells (Sagan, 1967; Margulis, 1970), are of microbial origin. It should be recalled, however, that tripolyphosphate and tripolyphosphatase are present in Rh. rubrum and mitochondria (Kulaev et al., 1972b; Konoshenko et al., 1973; Umnov et al., 1974b). It is logical to raise the question whether this enzyme, like adenosine triphosphatase and tripolyphosphatase, can or could participate in biosynthesis of tripolyphosphate coupled with electron transport. Having essentially lost the role of primary energy acceptors and donors in the course of evolution, high molecular-weight polyphosphates began to perform new functions. They play a particularly important part in the life of contemporary micro-organisms serving as pools of activated phosphate groups, high molecular-weight ion exchangers, and regulators of many metabolic processes. However, even in today’s micro-organisms, these compounds can be synthesized during glycolyticphosphorylation and utilized together with pyrophosphate in substrate phosphorylation (Kulaev, 1979). No matter how great was the role of polyphosphates and pyrophosphates in primitive micro-organisms, it appears that they were able to synthesize ATP prior to the development of photosynthesis or anaerobic respiration. If this were not so, modern fermenting bacteria could hardly have the capacity to synthesize ATP, as Broda (1971, 1975) indicates.

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Estimating the role of membranes in bioenergetic processes, Mitchell (1970) wrote: “It is an evolutionary attractive proposition that the proton-translocating oxidoreduction loop system and the reversible proton-translocating ATPase may have arisen separately as alternatives for generating the pH difference and membrane potential required for nutrient uptake and tonic regulation via porter systems in primitive prokaryotic cells and may then have provided the means of storing free energy of oxidoreduction in ATP synthesized by the reversal of the ATPase, or in some other anhydride, such as pyrophosphate, produced by a similar mechanism”. A very similar composition and identical structure of coupling membranes, high similarity of coupling mechanisms in membranes of photosynthetic and aerobic bacteria, as well as in mitochondria and chloroplasts, allowed Skulachev (1972b) to suggest that the system by which electron transport and phosphorylation are coupled, once created, was used in every organism that has survived until today without any fundamental change. This seems to hold true for energy-dependent synthesis of pyrophosphate, which occurs not only in chromatophores of very old micro-organisms from the evolutionary point of view, i.e. Rh. rubrum (H. Baltscheffsky et af., 1966; M. Baltscheffsky et af., 1966; Baltscheffsky and Stedingk, 1966) but also in chloroplasts of algae and higher plants (Rubtsov et af.,1976) and in mitochondria of lower and higher eukaryotic organisms (Mansurova et af., 1973a, 1975a,b, 1977a). It is very likely that, after the appearance of pyrophosphate synthesis coupled with electron transport, pyrophosphate synthesized in one way or another could, performing other functions as well, participate in maintenance of the electrochemical potential across the membrane (Skulachev, 1978). Evolution of bioenergetic processes also involved evolution and sophistication of regulatory systems. This could have led to supersession of high molecular-weight polyphosphates and pyrophosphate as monotonically built compounds by a more complicated multifunctional and readily recognizable structure, i.e. ATP. Nevertheless, even in mammalian cells, sufficiently high amounts of pyrophosphate (Guynn et al., 1974; Mansurova et al., 1977a; Veech et af., 1980) and certain quantities of polyphosphates (Kulaev and Rozhanets, 1973; Mansurova et af., 1975b) can be detected. Moreover, specific enzymes utilizing pyrophosphate in phosphorylation reactions have been identified (Nordlie and Arion, 1964; Stetten, 1964; Stetten and Tafft, 1964; Nordlie, 1976; Mansurova and Ibragimov, 1979). It is obvious that, in higher plants and animals, the importance of high molecular-weight polyphosphates and pyrophosphate in bioenergetic processes decreased. However, it is still unclear what specific functions these compounds have retained in the course of evolution from primitive forms of life to the highiy organized living beings of today.

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V. General Conclusions The present review devoted to the physiological role of inorganic polyphosphates was conceived with the purpose of depicting the precise state of investigations of this yet vague problem. A wealth of new data have appeared since publication of a detailed monograph (Kulaev, 1979) devoted to various aspects of the biochemistry of polyphosphates. Some findings provide certain support or, on the contrary, refute earlier assumptions and postulates, whereas others open new aspects in studies of inorganic polyphosphate metabolism. The general conclusion drawn from these data is that inorganic polyphosphates can play an essential part in metabolism of organisms containing these compounds, in the first place as high-energy phosphorus compounds functionally alternative to ATP. Moreover, the ever-increasing amount of data point to an important role of polyphosphates in regulation of numerous biochemical processes. A major achievement of recent investigations consisted in the formation of the concept about the heterogeneity of different polyphosphate fractions not only as regards chain length but also in both intracellular localization of the pathways of their biosynthesis and utilization and their functional role in the vital activities of the cell. Finally, it has become evident that studies on the biochemistry of inorganic polyphosphates contribute not only to the progress of fundamental science but also have practical significance for the most burning problem of the present time, namely preservation of the environment. Many problems of the biochemistry of polyphosphates have been raised in this review and necessitate further investigations. They are: 1. What are the precise mechanisms of the relationship between polyphosphate and nucleic acid metabolism? 2. Are there any specific mechanisms of polyphosphate transport from one membrane structure to others? 3. What are the relationships between the metabolism of high molecularweight polyphosphates and pyrophosphate? 4. What is the practical significance of polyphosphates as biological ion-exchangers?

We hope that these and many other prcblems of the biochemistry of polyphosphates will be successfully solved in the near future.

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V. Acknowledgements The authors are very grateful to their colleagues from the Laboratory of Regulation of Biochemical Processes of the Institute of Biochemistry and Physiology of Micro-organisms of the USSR Academy of Sciences and from the Department of Molecular Biology of the Lomonosov State University, Moscow, whose work on the biochemistry of polyphosphates in microorganisms stimulated writing of this review. Special thanks go to Drs S. E. Mansurova, M. A. Nesmeyanova, M. A. Bobyk, M. S. Kritsky, D. I. Nikitin, L. A. Okorokov and D. N. Ostrovsky for the kind submission of new data and for fruitful discussion. The authors’ thanks are also due to A. V. Mudrik and L. G. Sergeyeva for the assistance in the preparation of the manuscript and to V. D. Gorokhov for its translation into English. REFERENCES

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