The permeability properties of the lysosomal membrane

Biochimica et Biophysica Acta, 472 (1977) 419-449 © Elsevier/North-Holland Biomedical Press BBA 85177

THE

PERMEABILITY

PROPERTIES

OF THE

LYSOSOMAL

MEMBRANE

D I R K - J A N R E I J N G O U D and JOSEPH M. TAGER

Laboratory of Biochemistry, B. C, 19. Jansen Institute, University of Amsterdam, Plantage Muidergracht 12, Amsterdam (The Netherlands) (Received April 12th, 1977)

CONTENTS I.

General introduction

II.

Permeability of the lysosomal membrane to the products of hydrolysis . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Latency studies . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The sedimentability of lysosomal enzymes . . . . . . . . . . . . . . . 3. Morphological studies . . . . . . . . . . . . . . . . . . . . . . . B, The permeability to polyhydroxyl compounds . . . . . . . . . . . . . . C. The permeability to amino acids and small peptides . . . . . . . . . . . . D. The permeability to nucleosides . . . . . . . . . . . . . . . . . . . . . E. The permeability to salts . . . . . . . . . . . . . . . . . . . . . . . . F. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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421 421 421 424 425 425 427 428 428 430

III.

The intralysosomal pH: its magnitude and the mechanism of its regulation A, Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The meaning of pH . . . . . . . . . . . . . . . . . . . . . . 2. Methods for measuring the intralysosomal pH . . . . . . . . . . B. The magnitude of the intralysosomal pH . . . . . . . . . . . . . . C. The regulation of the intralysosomal pH . . . . . . . . . . . . . . 1. The ATP-dependent proton pump hypothesis . . . . . . . . . . 2. The Donnan type of equilibrium . . . . . . . . . . . . . . . . D. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . .

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431 431 432 433 435 438 438 441 442

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444

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445

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

445

IV.

General conclusions

Acknowledgements

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420

Abbreviations: CCP, carbonyl cyanide phenylhydrazone; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethane sulphonic acid; MES, 2-(N-morpholino)ethane sulphonic acid; MOPS, morpholinopropane sulphonic acid; DMO, 5,5'-dimetbyl-2,4-oxazolidinedione.

420 I. G E N E R A L I N T R O D U C T I O N

In 1955, de Duve and coworkers [1] proposed that rat-liver cells contain a distinct population of organelles which they called lysosomes. This proposal was based on biochemical and morphological data on 'dense bodies'. In particular, it had been observed that the behaviour in a centrifugal field of a group of hydrolytic enzymes in rat liver homogenate is different from that of mitochondrial enzymes, and that these hydrolytic enzymes exhibit structure-linked latency. Several years later, de Duve [2] introduced the concept of the vacuome, a system of intracellular vesicles in which two major intracellular processes take place, the synthesis and transport of secretory macromolecules and the digestion of intracellular and extracellular macromolecules. In one compartment of the vacuome, consisting of rough and smooth endoplasmatic reticulum and the Golgi apparatus, the synthesis and packaging of secretory macromolecules and of lysosomal enzymes take place. The former are packaged into secretory granules and the latter into primary lysosomes, lntra- and extraceUular material destined for lysosomal digestion is sequestered within phagosomes, which fuse with primary lysosomes to form secondary lysosomes. The secretory granules, the primary lysosomes, the phagosomes and the secondary lysosomes form the second compartment of the vacuome. The secretory granules can discharge their contents into the extracellular space following fusion with the plasma membrane (the process of exocytosis). Extracellular material can be taken up into the lysosomal system by invagination of the plasma membrane followed by the formation of heterophagosomes (the process of endocytosis). According to this model, the lysosomes comprise a dynamic system consisting of primary lysosomes, secondary lysosomes, heterophagosomes, autophagosomes (containing sequestered intracellular material) and, in addition, 'residual bodies', containing indigestible material. Thus an important feature of the lysosomal system is that it consists of heterogeneous elements, which poses problems when one studies the permeability properties of lysosomal membranes (see ref. 3 for a discussion of lysosomal heterogeneity*). The degradation of macromolecules in the lysosomes is catalysed by hydrolytic enzymes which, when isolated, have an acid pH optimum and exhibit little, if any, activity at neutral pH [4,5]. The functioning of the lysosomal system poses rather stringent requirements with regard to the characteristics of the matrix of the lysosome and of the surrounding membrane. Firstly, the low molecular weight compounds which are the products of the hydrolytic reactions must be able to leave the lysosomes. If this were not the case, an increase in osmotic value would occur as a consequence of the degradative processes, which could lead to swelling and bursting of the lysosomes. The products include monosaccharides [6J, amino acids and small peptides

* A distinction should be made between intracellular heterogeneity of lysosomes in vivo, and the heterogeneity of the particles in an isolated lysosomal preparation derived from a tissue containing more than one cell type.

421 [7], purine and pyrimidine nucleosides [8,9], phosphate, and sulphate. Furthermore, hydrolysis of macromolecules like proteins, nucleic acids and certain mucopolysaccharides yields products capable of releasing or binding protons. The observation that most of the hydrolytic enzymes have little, if any, activity at neutral pH's prompted Coffey and de Duve [7] to propose that the intralysosomal pH must be low. Furthermore, there must be a mechanism by means of which the intralysosomal p H is kept within certain limits, especially if acid-base equivalents are produced. Accordingly, two prerequisites for the occurrence and continuation of intralysosomal hydrolytic processes can be formulated: the maintenance of the proper intralysosomal pH and the maintenance of a constant osmotic value within the lysosomes. The purpose of this article is to review the literature on the permeability properties of the lysosomal membrane in relation to these prerequisites. (For recent reviews see refs. 10-13; see also ref. 14).

II. PERMEABILITY OF THE LYSOSOMAL MEMBRANE TO THE PRODUCTS OF HYDROLYSIS

IIA. Introduction The heterogeneity in the lysosomal population implies that the permeability properties of the lysosomal membrane studied in cell-free preparations represent the average properties of the whole population. The methods mainly used in studying the permeability properties of the lysosomal membrane involve measurement of either the latency or the sedimentability of lysosomal enzymes after suspension of a lysosomal preparation in solutions of low-molecular weight compounds. The advantage of these methods is that a rather crude lysosomal preparation can be used, so that the problem of purification of lysosomes can be circumvented [16]. 1. Latency studies. Lysosomes, like other vesicular membrane systems, swell when they are suspended in isotonic solutions of a permeant solute (see ref. 15). This type of swelling is essentially the movement of water into the vesicles following the increase in internal osmolarity. The swelling can lead to rupture of the vesicles. If the lysosomal membrane is impermeable to the substrate of a lysosomal enzyme, no activity of the enzyme can be detected in the intact lysosomes: the enzyme is latent*. The activity can be unmasked by lysing the lysosomal membrane [17,18]. Thus the rupture of the lysosomal membrane induced by swelling, and hence the permeability to a solute, can be monitored by measuring changes in the latency * In this review the lysosome is considered to be a membrane-bound vesicle. In our opinion, there is no convincing evidence for the matrix-binding theory advanced by Koenig [42] as a model for the structure of the lysosome.

422 of a lysosomal enzyme after suspension of the lysosomes in solutions of that solute. The possibility exists that the lysosomal membrane may not be completely impermeable but may have a restricted permeability to the substrate. A distinction between these two possibilities can be made by studying the dependence of the latency on substrate concentration. If restricted permeability for the substrate exists, the latency should decrease at higher substrate concentrations. If the membrane is virtually impermeable, the latency is independent of the concentration of substrate used. In his discussion of structure-linked latency, de Duve [17] stressed that the latency of acid phosphatase is independent of the concentration of fl-glycerol phosphate used in the assay of the enzyme [19]. Both free and total activity exhibit the same affinity for the substrate (the Km value is the same), and differ only in the amount of enzyme activity found in the preparation before and after lysis (Vis greater after lysis). The most probable explanation is that the membrane does not show any measurable permeability to fl-glycerol phosphate; if substrate penetration were ratelimiting, a change in Km would be expected. This is also clear from the observation that fl-glycerol phosphate offers good osmotic protection of the lysosomes. Similar results have been obtained with three other lysosomal enzymes. When the activity of/q-glucoronidase [20], hexosaminidase [21] and acid a-glucosidase [22] is measured in intact and lysed preparations using the p-nitrophenyl glycosides as substrates the same Kr~ but different V values are found, indicating again that the membrane is impermeable to the substrates. To extrapolate these observations to all lysosomal enzymes and to all substrates for one enzyme may lead to difficulties. Indeed, there are a number of reports in the literature in which it is suggested that the lysosomal membrane is permeable to some substrates used for the assay of hydrolases [23-31]. In the case of fl-galactosidase, Baccino and Zuretti [20] reported that both V and Kr, differ in an intact and in a lysed rat-liver homogenate. However, Burton and Lloyd [22] found that the Km values of free and total fl-galactosidase are the same when measured in a mitochondrial-lysosomal fraction. The possibility exists that a non-lysosomal fl-galactosidase may be present in the crude homogenate used by Baccino and Zuretti [20]; the contribution of such an isoenzyme may predominate when 'free' activity is measured. A non-lysosomal fl-galactosidase has been reported in rat kidney [32] and may also be present in rat liver. Furthermore, the results of Burton and Lloyd [22], show that the latency of a particular lysosomal enzyme may differ depending on the substrate used. They showed that the Km value for ct-fluoroglucoside of a-glucosidase in an intact preparation differs from that in a lysed preparation, whereas the same Km value is found in the two types of preparation when maltose or p-nitrophenyl-a-glucoside is used as a substrate [22]. In many cases, however, the data are insufficient to allow firm conclusions to be drawn concerning restricted permeability of the lysosomal membrane to the substrate used. An exception to the general impermeability of the lysosomal membrane towards the substrates of lysosomal hydrolases is also formed by certain dipeptides, tripeptides

423 and other compounds, as indicated by the studies of Goldman and coworkers [28-30]. They have found that addition of several of these compounds to a suspension of lysosomes in isotonic sucrose leads to lysis of the lysosomes, as measured by the decrease in latency of acid phosphatase. Goldman and coworkers [28-30] suggest that the compounds enter the lysosomes and are hydrolysed inside, and that the hydrolytic products permeate only slowly out of the lysosomes. As a consequence, the osmotic value increases and the lysosomes swell and subsequently burst. In such cases, the increase in osmotic value in the matrix is determined by the rate of permeation of the substrate (in itself a function of the concentration gradient across the membrane), the rate of hydrolysis of the substrate in the lysosomes, and the rate of effiux of the products. The results published by Goldman and coworkers [28-30] and, more recently, by Bouma et al. [31], are too limited to allow a detailed examination in terms of permeation coefficients of the substrates and hydrolytic products. Firstly, only limited kinetic data are given. Secondly, the exact localization of the enzymes involved is not well documented in the studies of Goldman and coworkers; this is of particular importance in the case of the lysis induced by the O-methyl and O-ethyl esters, since methanol and ethanol, the hydrolytic products, are membranedisrupting agents. On the other hand, Bouma et al. [31] have shown clearly that the dipeptidases for Leu-Gly and Ile-Gly are located in the lysosomes. If latency represents the percentage of intact lysosomes, the decrease in latency with time observed under particular conditions represents a cumulative effect. If the amount of osmotically active material in the lysosome (and/or their diameter) shows a normal distribution, the curve describing the time course of the decrease in latency (expressed as free activity and plotted versus time) in experiments in which lysosomes are suspended in hyperosmotic solutions of a (slightly) permeable solute will be S-shaped (cumulative normal distribution). The time taken to reach half-maximal decrease in latency will be a function of the rate of permeation of the solute. Furthermore, if the medium contains an impermeant solute and is hypoosmotic the curve describing the time course of the decrease in latency (free activity versus time) should in principle also be S-shaped. In this case, the maximum decrease in latency and the time required to reach half-maximal decrease in latency will depend on the concentration of osmotically active species in the medium. The curve relating the maximal decrease in latency to the concentration of impermeant solute (percentage latency plotted against concentration) might be expected to be S-shaped. However, in experiments published thus far in which the latency of lysosomal enzymes has been followed in time in the presence of permeable compound no S-shaped curves have been obtained (e.g. see ref. 21). Furthermore, the published curves relating free activity to concentration of impermeant solute do not have a pronounced S shape (e.g. see ref. 33). This problem has recently been reexamined in our laboratory (M. Hollemans, unpublished observations). If a mitochondrial-lysosomal fraction is isolated in a hyperosmotic solution (500 mM sucrose) and transferred to solutions of lower osmolarity, and if the latency is plotted as a function of concentration of sucrose, a

424 EFFECT OF OSMOLARITY OF MEDIUM ON LATENCY OF ACID PHOSPHATASE 100

r

r

75

- - 50 ,

8

0

.

100

200 300 Sucrose (mM)

4;0

500

Fig. l. Effect of osmolarity of medium on latency of acid phosphatase. A crude mitochondriallysosomal fraction from rat liver was isolated as described by Reijngoud et al, [106] except that the isolation medium contained 500 mM sucrose instead of 250 raM. This fraction was suspended in media containing 10 mM MES, 10 mM MOPS, sufficient Tris to adjust the pH to 7.5 and sufficient sucrose to give the indicated final concentration. To obtain the low final concentrations of sucrose the fraction was diluted. After incubation for 90 rain at 0°C, samples were removed and suspended in the reaction mixture for acid phosphatase described in ref. 1, except that the sucrose concentration was 500 raM. Acid phosphatase was determined as described as in ref. 1. curve with a pronounced S shape is obtained (Fig. !). In most published experiments the lysosomes have been isolated in 250 m M sucrose, so that in experiments o f this type only part of the curve o f Fig. l is seen, In order to be able to transform the data obtained in latency experiments into permeability coefficients, a systematic and detailed study of the latency p h e n o m e n o n is essential, utilizing the principles o f irreversible thermodynamics (see ref. 34-36). In this respect it is o f importance to note that the latency experiments bear much resemblance to the haemolysis experiments o f erythrocytes [37-39]. A n o t h e r problem in the latency measurements is the possible difference in distribution of the enzymes a m o n g the subpopulations in a lysosomal preparation (see Section IIA). Although the original data o f de Duve et al. [40] show that the latency o f different enzymes decreases in a similar way after treatments designed to disrupt the lysosoma] membrane, it should be borne in mind that such methods are too insensitive to be able to distinguish between different populations o f lysosomes (see also ref. 41). This problem needs further study (see ref. 22 for a discussion). As may be inferred from the preceding discussion, latency of an enzyme can be used to measure the rate o f lysis induced by solutes if the substrate used is nonpermeant. It should be pointed out that the presence o f c o m p o u n d s causing changes in the lysosomal m e m b r a n e m a y lead to alterations in the permeability to solutes. Such changes can be brought about by incubating lysosomes with polyelectrolytes [44--46] or drugs [47,48] or by extreme pH values [42,43]. 2. The sedimentabiBty of lysosomal enzymes. The second method o f studying

425 the permeability properties of the lysosomal membrane makes use of the following rationale. If the lysosomes burst because of osmotic imbalance, the enzymes are released into the medium. Using centrifugal techniques it is possible to separate particle-bound enzyme activity from free activity. The particle-bound fraction is taken to represent the unbroken lysosomes. However, there are several objections against this method. Firstly, the sedimentable enzyme activity represents not only that contained in unbroken lysosomes but also that bound or adsorbed to membrane fragments. Secondly, several authors [46,48-62] have shown clearly that not only the osmotic value but also the ionic strength and composition of the medium are of influence on the sedimentability of enzymes. Thirdly, it appears that this method is not as sensitive to swelling-induced breakage as the latency measurements; often, a decrease in latency is not accompanied by a decrease in sedimentability [38,46,48,49,52,60]. Finally, the partition of the activity between the membrane and the soluble phase depends on the enzyme studied [63-73]. Thus sedimentability measurements, especially when used to compare different incubation conditions, are very difficult to interpret. 3. Morphological studies. In one respect the latency and the enzyme sedimentability methods give the same type of information, since in both we are concerned with the influx of compounds into the lysosomes. In the in vivo situation the predominant flow of low molecular weight compounds is from the inside to the outside of the lysosomes. Accordingly, a few studies [74-79] have been carried out in which the in vivo situation is imitated. Non-toxic, indigestible compounds like sucrose are injected into intact animals [78,79] or added to the medium of cultured cells [74-77]; these compounds are subsequently taken up by the livers of the intact animals or by the cultured cells, presumably by bulk endocytosis [78]. Large, swollen vacuoles are formed if the compounds are non-permeants. If digestible compounds are used, and if lysosomal breakdown of these compounds can be demonstrated, lack of vacuolization may indicate that the lysosomal membrane is permeable to the products of hydrolysis; this is the case with maltose, isomaltose, melibiose and lactose [77]. This method gives only a qualitative indication of the permeability.

liB. The permeability to polyhydroxyl compounds The permeability of the lysosomal membrane to different polyhydroxyl alcohols has been tested in a light mitochondrial fraction of rat liver [21,80-83], in homogenates of Tetrahymena pyriformis [84-86], and in a system in which enlargement of lysosomes within endocytosing cells is used as a measure of the permeability of the membrane to the endocytosed compounds [74-76]. The most extensive study has been that of Lloyd using rat-liver lysosomes [21]. In his experiments he used the decrease in latency of arylsulphatase as a marker for the permeability to polyhydroxyl compounds. He showed that there is a progressive decrease in permeability of the lysosomal membrane to these compounds, depending in general on their size and charge, as follows: glycerol > tetritols > pentoses

426 hexoses > pentitols > hexitols = inositol = disaccharides = oligosaccharides ~charged sugars and lactate*. The oligosaccharides, disaccharides, hexitols and charged sugars all gave good osmotic protection, indicating that they are relatively impermeant. The charged saccharides tested were gluconate and glucuronate. The 7-1actone of glucuronate was as permeable as galactose. Badenoch-Jones and Baum [82] have found the same selective permeability. The results of a study using a homologous series of polyethylene glycols led BadenochJones and Baum [82] to conclude that the molecular weight is an important factor determining the permeability of the lysosomal membrane to these compounds. Polyethylene glycols with a molecular weight of 1000, 600 or 400 were impermeant, whereas those with a molecular weight of 200 were about half as permeant as glycerol. Hainsworth and Wynn [81] have also shown that glycerol is permeant, in contrast to sorbitol (a hexitol). In a study of the permeability properties of the lysosomal membrane of Tetrahymena, Lee [84-86] showed that incubation in sucrose, raffinose or melibiose led to only a slight decrease of latency, indicating impermeability of the lysosomal membrane to these compounds. Glucose, a-methylglucose, galactose, sorbitol and mannitol were permeant to about the same degree, whereas glycerol was more permeant than the monosaccharides and their derivatives. All the charged monosaccharides tested (glucuronate, gluconate and glucosamine) were only slightly permeant. When macrophages are suspended in carbohydrate-containing solutions, vacuolization of the lysosomes is induced by the disaccharides trehalose, cellobiose, sucrose, gentiobiose and turanose, the trisaccharide raffinose, and the tetrasaccharide stachoyose, indicating that the rate of endocytosis is greater than that of the efflux of the compounds from the lysosomes [77]. In contrast, lactose, melibiose, maltose, and isomaltose do not induce extensive vacuolization, presumably because they are broken down by the hydrolytic enzymes of the lysosomes to the permeant monosaccharides glucose and galactose [77]. Nyberg and Dingle [76] also showed that glucose is permeant, but found that lactose leads to extensive vacuolization. The difference in response to lactose in the experiments of Cohn and Ehrenreich [74] and Nyberg and Dingle [76] may be due to the presence of a lactose-metabolizing enzyme either in the macrophages or in the medium used by Cohn and Ehrenreich [74]. When sucrose- or cellobiose-loaded cells are incubated with fl-fructofuranosidase or fl-glucosidase, respectively, the extensive vacuolization disapperars indicating a permeability of the lysosomal membrane for fructose and glucose [74]. The general conclusion from these qualitative results is that the lysosomal membrane is relatively permeable to monosaccharides but not to disaccharides or trisaccharides. Although the published data do not allow definite conclusions to be drawn about the presence or absence of specific carrier mechanism(s) for the transport of * The osmotic protection afforded by lactate is probably due to impermeability to the cation (see Section liE). Most biological membranes and artificial phospholipid membranes appear to be relatively permeable to monocarboxylic acids (see ref. 87).

427 polyhydroxyl compounds across the lysosomal membrane, Lloyd's [21] observation that the permeability of the lysosomal membrane for uncharged polyhydroxyl compounds decreases as the size of the molecule increases, with no clear-cut break between mono- and oligosaccharides, argues against the existence of a specific uptake mechanism in these organelles. Stein [88] and also Diamond and Wright [89] have suggested that the main factor determining the rate of permeation of a non-electrolyte through a membrane is the passage of the molecule through the membrane/water interface, this, in turn, being determined by the rate of desolvation of the compound before it can enter into the membrane phase. In addition, solute-lipid interactions are important, as can be deduced from results with artificial membrane systems [90-93] and from the selectivity pattern of the permeability of the mitochondrial membrane for aldoses and polyhydroxyl compounds [94]. The behaviour of charged and uncharged monosaccharides is completely different. This can be the consequence of their charge and is then in accordance with observations that the lysosomal membrane is, in general, relatively impermeable to cations and anions (see Section IID). However, the influence of the charge of moncsaccharides has only been tested by studying the influx of these compounds into the lysosomes, using osmotic protection as a criterion of impermeability. No experiments have been performed in which the influence of charge on the efftux of these compounds out of the lysosomes has been studied. This could perhaps be done by using the technique described by Cohn and Ehrenreich [74] and Jacques [79]. The cells can first be allowed to endocytose indigestible non-toxic oligosaccharides containing the charged monosaccharide and subsequently to take up the hydrolase(s) required to degrade the oligosaccharide. Abolishment by the hydrolytic enzyme(s) of the vacuolization induced by the oligosaccharide would indicate that the lysosomal membrane is permeable to the charged monosaccharide. IIC. The permeability to amino a~ids and small peptides Lloyd [95] has studied the permeability of the lysosomal membrane to amino acids and small peptides, using the ability of these compounds to afford osmotic protection as a measure of their permeability. In general, amino acids have been found to be relatively impermeant. However, the permeability for amino acids depended on the pH of the medium. Thus glutamate and lysine provided greater osmotic protection at pH 7 than at pH 5 or 6, whereas glycine and alanine were relatively impermeant at all three pH levels. It should be pointed out that the degree of ionization of glutamate and lysine is much greater than that of glycine, alanine and valine in the pH range 5-7, so that the former two compounds should be considered as ionized salts. Indeed the concentration of Na + or CI- in the incubation medium containing 0.25 M glutamate or lysine was approximately 0.25 M, whereas for the other amino acids it was below 0.01 M [95]. Since the lysosomal membrane is relatively impermeable to Na + (see Section IIE) or C1- (Casey, R. E., unpublished

428 observations), suspension in 0.25 M glutamate or lysine will provide osmotic protection even if the amino acids are permeant. Lee [96] reported that the lysosomal membrane of Tetrahymena pyriformis is permeable to a series of amino acids. However, since he did not define the medium pH, it is impossible to compare his results with the data obtained with rat liver lysosomes. The permeability of the lysosomal membrane towards small peptides has been studied by the same technique [95,96]. However, the results cannot be interpreted unequivocally, since Goldman [29] and Bouma et al. [31] have shown that some dipeptides may be hydrolysed within the lysosomes after permeating through the membrane. If degradation of the dipeptides leads to formation of relatively impermeant products, an additional increase in the internal osmotic value will be obtained, leading to an overestimation of the rate of entrance of the compound and thus of its permeability coefficient. Alternatively, if relatively permeant products are formed there may be no change in the internal osmolarity so that an apparent osmotic protection is seen. It is thus of importance to test whether or not degradation takes place. Although several reports [97-99] have been published on the presence of dipeptidase activities within the lysosomes not all activities are known. Ehrenreich and Cohn [77] have investigated the ability of small peptides to induce vacuolization in endocytosing cells. Only (o-Ala)3 and (o-Glu)2 were able to induce vacuolization, whereas (D-AIa)2, D-Ser-D-Ala, D-Val-D-AIa, Gly-D-AIa, D-Ala-o-Thyr and D-Arg-D-Thyr had no effect. None of the peptides was broken down by cell lysates or by the calf serum present in the medium. It thus appears that the latter six peptides are able to permeate through the lysosomal membrane.

liD. The permeability to nucleosides Only one report has been published on the permeability of the lysosomal membrane to nucleosides. Using latency measurements Burton et al. [I00] concluded that the permeability of the lysosomal membrane to pyrimidine nucleosides is as follows: cytidine < uridine < thymidine. The permeability to uridine is significantly higher than that to glucose. Purine nucleosides seem to damage the lysomal membrane by interference with the membrane structure [100]. liE. The permeability to salts There is a discrepancy in the literature about the ability of salts of small ions like KCI and NaCI to afford osmotic protection to lysosomes. Early reports from the group of de Duve and coworkers [19,101] showed that of the salts tested, KC1 and NaCI gave only slight osmotic protection, in contrast to MgCI2, CaCI2, Na2SO4 and sodium acetate, which gave a protection comparable to that of sucrose. On the other hand, the results of Lloyd [95] and of Verity and Brown [102] suggested that the lysosomal membrane is impermeable to KC1 and NaCI. An explanation for this apparent discrepancy is given by the observations of Davidson and Song [103]. They found that when lysosomes isolated from kidneys of mice previously injected with

429 125I-labelled ribonuclease were incubated at 0 °C in a medium containing KC1, the radioactive ribonuclease could not be sedimented. In contrast, the radioactivity was sedimentable if the incubation was carried out in the same medium at 37 °C. This important result demonstrates that the change in the incubation temperature from 37 and 0°C induces a dramatic change in permeability properties of the lysosomal membrane. Whereas the lysosomal membrane is relatively impermeable to salts at 37 °C, it becomes relatively permeable at 0°C. Although the conclusions of Davidson and Song [103] are qualitatively correct, the use of sedimentability of a digestible macromolecule as a quantitative criterion of intactness of the lysosomal membrane introduces difficulties. For instance, in the case of lysosomes suspended in sucrose the higher sedimentability o f ribonuclease at 0°C than at 37°C [103] could be due either to an increased permeability to sucrose at 37°C in comparison with 0°C, a decreased breakdown of ribonuclease at 0°C in comparison with 37°C, or both. On the other hand, the decrease in sedimentability in a salt solution at 0°C in comparison with 37 °C is an underestimation of the difference in permeability to salt at the two temperatures, since at 0 °C breakdown of ribonuclease is low. It should be pointed out that the complications introduced by intralysosomal hydrolysis of macromolecules in analysing lysosomal breakage is discussed by the same group in another context [103]. Thus a difference in permeability properties at different temperatures may be the explanation for the discrepancy between the results of de Duve and coworkers [19,101], who performed their experiments at 0°C, and Lloyd [95] and Verity and Brown [102], who used a temperature of 20 or 37°C, respectively. Furthermore, this may also be the explanation for the early observation of Bertini et al. [50] that the sedimentability of endocytosed 125I-labelled albumin was lost when the isolated lysosomes were incubated in a salt solution at 0°C. These changes in permeability of the lysosomal membrane towards salts are not observed with MgC12 and CaC12, which afford good osmotic protection both at 0°C and at 37 °C. The question arises of whether the slow permeation of monovalent cations observed at 37°C occurs in vivo as well as in vitro. It is necessary to distinguish between an intrinsic, slow permeability to monovalent cations and a permeability due to changes in the lysosomal membrane arising during incubation of the isolated lysosomes. These changes could perhaps be brought about by enzymes derived from damaged lysosomes. This kind of in vitro change has been observed in mitochondrial preparations [104] which, of course, contain lysosomes. Indeed, a method of preparing lysosome-free mitochondria has been described in order to prevent damage to the mitochondria [105]. The investigation of Verity and Brown [102] on the permeability of the lysosoreal membrane to ammonium phosphate and acetate deserves special mention. These authors compared the loss of latency of hexosaminidase in lysosomes suspended in solutions of KC1, ammonium phosphate and ammonium acetate and concluded that the lysosomal membrane is permeable to acetate, relatively permeable to phosphate and relatively impermeable to KC1. However, examination of the time

430

membrane

out Ac- ~

HAc

in

DH A c ~ - ~ AcH+

NHZ: /

~NH3 H20

. NH3*-~-~ NH~ -~H20

Fig. 2. Mechanism of ammonium acetate-induced swelling in vesicular membrane systems. A c denotes the dissociated and HAc denotes the undissociated form of acetic acid.

course of the loss of latency (Fig. 6B of ref. 102) indicates that these conclusions are not entirely correct. In KCI, the rate of loss of latency was approx. 0.6 ~ per rain. In ammonium acetate the rate of loss of latency was approx. 3.5 ~ per rain during the first 5 rain and decreased continuously between 5 and 20 rain. In the case of ammonium phosphate, there was an initial rapid phase (approx. 3.5 ~ per rain) during the first 2 rain followed by a phase in which the rate was approximately equal to that observed in KCI. Assuming that the lysosomal membrane, like other biological membranes, is permeable to the uncharged form of weak acids and bases, it can be expected that the lysosomes will take up ammonium acetate (see Fig. 2) and that they will therefore burst. The situation is different with ammonium phosphate. Due to the acidic interior of the lysosomes (see Section llID) ammonia will rapidly accumulate in the lysosomes [106], leading to an initial rapid increase in osmotic value. The subsequent slow rate of loss of latency is apparently due to a slow permeation of ammonium ions together with phosphate ion, analogous to that of KCI, Thus it can be concluded that the lysosomal membrane, like other natural and artificial phospholipid membranes, is readily permeable to ammonia and acetic acid, and relatively impermeable to K +, CI- and phosphate ions. In some membranes, of course, specific transport systems for CI- or phosphate are present; this transport is electroneutral. There is no evidence for such transport systems in the lysosomal membrane. IIF. Conclusion

From the available data on the permeability properties of the lysosomal membrane, as surveyed above, no clear-cut picture emerges with regard to the mechanism(s) of permeation across the lysosomal membrane of the products of the hydrolytic reactions occurring within the lysosomes. In most studies of the permeability of the lysosomal membrane, the influx of solutes from the medium into the lysosomes is measured, at a pH of 7.0-7.5. However, under physiological conditions efflux of the products of intralysosomal hydrolysis occurs from the lysosomes, where the pH is relatively low (see Section II1). Since the degree of ionization of the permeant strongly depends on the pH of the medium, the in vitro studies do not necessarily yield information about the rate at which the lysosomes release their products of hydrolysis.

431 It should be borne in mind that during hydrolysis the lysosomes remain intact, so that removal of the hydrolytic products must occur. However, a high permeation rate of the products may not be obligatory and a low rate of diffusion may be sufficient to keep up with the rate of formation of the products (see ref. 13, for a discussion). At present, only qualitative conclusions can be drawn with regard to carbohydrate and polyhydrc,xyl compounds. It appears that there is a continuous increase in permeability as one goes from oligosaccharides to monosaccharides, that the hexitols are impermeant, and that glycerol is permeant. In the case of electrolytes, like charged carbohydrates, amino acids and salts, the data are too scattered to draw any firm conclusions. Furthermore, a phase transition probably occurs in the lysosomal membrane between 20 and 0 °C [103,153]. At 0 °C the membrane becomes relatively permeable to small cations and anions like K +, Na ÷ and CI-, whereas at 20°C or higher the membrane appears to be relatively impermeable to these ions.

Iti. THE INTRALYSOSOMAL pH: ITS MAGNITUDE AND THE MECHANISM OF ITS REGULATION IliA. Introduction During the lysosomal breakdown of certain macromolecules like nucleic acids and proteins, acid-base equivalents are set free. This may result in an alteration in intralysosomal pH, and may lead to a change in the relative contribution of different enzymes to the degradative processes, as proposed by Goettlich-Riemann et al. [107]. Alternatively, a mechanism may exist to prevent alterations in pH. Intimately bound up with this problem is the question of the magnitude of the intralysosomal pH. As already pointed out in the general introduction (Section I), the lysosomal enzymes have an acid pH optimum, and most of them exhibit little, if any, activity at pH levels near neutrality. This observation led Coffey and de Duve [7] to propose that the intralysosomal pH must be low in comparison with that of the surrounding medium. If this is the case, the problem arises of how a low intralysosomal pH is achieved and how it is maintained and regulated. Two general mechanisms have been proposed. The first is an energy-dependent mechanism in which it is visualized that protons are actively pumped into the lysosomes, the energy being provided either by a redox reaction [4] or by ATP [108]. According to the second mechanism a Donnan-type equilibrium is involved. If proton transport into the lysosomal matrix is directly coupled to a redox reaction or an ATPase present in the lysosomal membrane, an electrochemical gradient of protons will be built up which will finally be in equilibrium with the energy-delivering reaction and thus stop further pumping of protons, unless there is a concomitant, charge-compensating flux across the lysosomal membrane. This charge-compensating flux could be either an influx of anions into the lysosomes or an efflux of cations from the lysosomes.

432 According to the scheme based on a Donnan-type equilibrium [10%115] a pH difference across the lysosomal membrane (acidic inside) is brought about by the presence of indiffusible or fixed negatively-charged groups within the lysosomes. This is only possible if the membrane is permeable to acid-base equivalents and if their permeation is electroneutral. In the following sections, the meaning of p H and the methods used to estimate intralysosomal p H will be discussed. The final sections will be devoted to a discussion of the regulation of intralysosomal p H in relation to the two general mechanisms outlined above. 1. The meaning ofpH. Before discussing the methods for measurement of the intralysosomal p H and the results obtained with these methods, it is of importance to discuss briefly the concept of p H (for more extended reviews, see refs. l l 6-119). The p H of a solution is defined as: pall = - - log aH

(l)

in which an = activity of protons, and a~ = 7a C.

(2)

where ;e. = activity coefficient of protons, and CH = concentration of protons. The application of this definition to an actual situation leads to fundamental problems. The acidity of the medium, conventionally called pH, can be measured only indirectly, either by means of an indicator dye or electrochemically. In the former method use is made of the fact that the dye exhibits an acid-base equilibrium and that the protonated and unprotonated forms exhibit a colour difference. For an acid-base equilibrium in a solution the Henderson Hasselbalch equation holds: pan = pK~ ÷ log al_

(3)

an!

in which arn = activity of the protonated species, af = activity of the unprotonated species, and pK~ = negative logarithm of the acid-base dissociation constant of the indicator. However, the colour of the indicator dye, measured spectrophotometrically yields information about the ratio of the concentrations of the different indicator species, and not of aJam. The relationship between pan as defined in Eqn. 3 and the measured quantity (pH) can be derived as follows: Since pan = pK~ + log al ,

(4)

all!

a.i = 7m Cr,

(5)

and al

= 7J CI

where 7m =

(6)

activity coefficient of protonated species, Cm = concentration of

433 protonated species, 7~ = activity coefficient of unprotonated species and C~ = concentration of unprotonated species, it follows that pall = pK~ + l°gCllt.H Yt Vm

(7)

pH = pall - - log 7 I 7m

(8)

pH = pKa + l o g ~

(9)

Thus, to evaluate the pall, it is necessary to know the activity coefficients of HI and I at the pH of the medium. However, there is no feasible method of measuring the pan independently of Ym and 8i. The electrochemical method, too, yields no exact information about the paw The uncertain factor in the relationship between the pax and the potential difference between the electrodes appears to be the liquid junction potential across the salt bridge connecting the calomel electrode with the solution [120]. The magnitude of the liquid junction potential is a function of the composition of the medium and thus a function of the paw The same paradoxical situation arises as with the dye method. Both methods indicate a pH, in part determined by the pax, but also by the uncertainties specific for each method. The differences in the uncertainties characteristic for each method will lead to a discrepancy in the p H indicated by the two methods. These discrepancies have been well documented in the early literature concerning pH measurements [121-134]. They were thought previously to be due solely to an error in the pH estimated by the dye method [121]. Different indicators give different estimations of the pH, under the same experimental conditions. The reason for this phenomenon is that the indicators are organic molecules differing in sensitivity to ionic strength (which can lead to a kind of 'salting-out' of the uncharged species), in degree of binding of ions, and in degree of absorption to proteins and other macromolecules present in the solution ('protein error') [125-127, 134]. For a discussion of the effect of these factors, in particular the 'protein error', the reader is referred to refs. 113 and 135-138). In this respect, it is of importance to mention that the electrochemical determination of the pH is also sensitive towards the presence of polyelectrolytes (for a discussion, see ref. 93). In conclusion, the measured p H of a solution is only an operationally defined quantity, which is obtained by extrapolation using buffers of standard pH as the reference. The extent to which the quantity thus obtained is actually determined by the acidity of the medium depends on the experimental circumstances. Furthermore, no transformation can be made in which the concentration of protons can be calculated from the measured pH. In any case, the participation of protons in chemical and biochemical reactions is expressed in terms of the electrochemical potential, //H+, which is a function of the logarithm of the proton activity, and not of the proton activity itself [139]. 2. Methods for measuring the intralysosomal pH. The methods that have been

434 employed to measure the intralysosomal pH are based on the colour changes of endocytosed, particle-bound acid-base indicator dyes [140-150], the distribution of radioactively labelled lipid-soluble weak bases and acids across the lysosomal membrane [106,151-155] and the response of an intralysosomal degradation process to the incubation conditions in vitro [106,151,156,157]. In the first method, endocytosing cells are incubated with a particle-bound dye and the colour of intraceHularly located dye is compared with that of particle-bound dye in standard buffers. So far this method has been employed for the determination of the intralysosomal pH in intact ceils like macrophages [140-146] and in unicellular organisms containing food vacuoles [147-150]. No experiments have been reported in which colour changes have been measured in response to experimental conditions in isolated lysosomes filled with matrix-bound indicator. Besides the uncertainties in the pH determinations themselves mentioned in Section lIIb, there are other uncertainties as well, so that this method gives only a rough estimation of the intralysosomal 'pH'. Firstly, the estimation of the colour is done by eye and not spectrophotometrically. Secondly, the dye is complexed to the matrix by boiling the particles, for instance yeast cell walls, with the indicator for several hours. It is possible that subsequently redistribution of the dye within the lysosomes occurs, and that a part becomes bound to the membrane of the lysosomes (see discussion following ref. 18). The second method for calculating the intralysosomal pH is based on distribution measurements of radioactively labelled lipid-soluble weak bases or acids. Isolated lysosomes are suspended in a medium containing the indicator compound. After equilibration of the compound across the lysosomal membrane the lysosomes are centrifuged down, the radioactivity in both pellet and supernatant is measured, and the accumulation factor, in terms of concentrations, is calculated. To transform this accumulation factor into d p H , Eqn. 9 is applied on both sides of the membrane. Several assumptions have to be made [I58-161]. Firstly, it is assumed that the membrane is permeable to the uncharged species only and impermeable to the charged species of the indicator compound. Secondly, it is assumed that no additional binding to lysosomes occurs by mechanisms other than protonation or ionization of the base or acid in the matrix of the organelle. Finally, it is assumed that the pK~ of the indicator compound is the same within the vesicle as in the medium. For a weak base as indicator compound the following relationship holds : phi, -- pK~- log(f(l + 10P•a-Pno)--1)

(I0)

in which pHi, = pH at the inside of the vesicle, pHo = pH outside the vesicle and f ~= concentration factor of the indicator compound at the inside of the vesicle, whereas for a weak acid the relation is pHi, - pK~ ~- log(f(l i 10pno-v•a)-l)

(11)

The rationale of the third method, in which the intralysosomal pH is calculated

435 from the rate of an intralysosomal degradation process, is based on experiments in which the effect of attaching enzymes to polyelectrolytic matrices on the rate of the reactions catalysed by these enzymes as a function of medium pH has been studied [162-164]. Using matrix-bound enzymes, the pH difference between the bulk phase and the matrix phase can be calculated from the enzyme activity. This pH difference agrees with that expected on the basis of the charge density of the matrix. In the case of lysosomes the experiments are performed in the following way. Isolated lysosomes, containing endocytosed 125I-labelled protein, are incubated at different medium pH levels, and the rate of degradation of protein as a function of medium pH is determined. Subsequently, the same degradation process, but now catalysed by lysed lysosomes, is monitored at different medium pH levels. Both curves are normalized, the activity at the pH optimum being taken as 100 ~. The pH curve of the degradation process in intact lysosomes is displaced in comparison with that obtained with lysed lysosomes, indicating that a pH difference across the lysosomal membrane exists. The magnitude of this ApH is calculated by extrapolation (see ref. 106). The main objections against this method is that no experimental evidence is available indicating that the kinetic parameters of the degradation process are the same in intact and in lysed lysosomes. 'Furthermore, the stability of the lysosomal membrane can also interfere with determination of the intralysosomal pH by this method, since breakage of the lysosomes will lead to the termination of the hydrolytic process because of dilution of substrate. Davidson [165] and Davidson and Song [103] have attempted to distinguish between these two factors by means of a kinetic analysis. However, it is not clear how a distinction can be made between the kinetic constants of these two rate-determining factors, if they occur simultaneously, particularly since Davidson and Song [103] use the decrease in sedimentability of the radioactively labelled protein as a measure of lysis. To overcome this difficulty it would be necessary to use an inhibitor of the degradation process, so that the sedimentability is really a measure of lysis. All three methods for determining the intralysosomal pH have their specific uncertainties and all three are based on certain assumptions. In our opinion, reliable information can be obtained only by comparing the data obtained with different methods.

IIIB. The magnitude of the intralysosomal pH The measurements of the value of the intralysosomal pH in phagocytosing cells using matrix-bound indicator dyes are summarized in Table I. These results show clearly that a relatively low pH exists in the lysosomes in the cell. However, the estimated values within one cell type depend on the indicator dye used. This is especially clear in Mandell's [143] experiments. The discrepancies observed must reflect interactions of the bound indicator with the lysosomal matrix other than those due to pH, such as a difference in sensitivity to protein or in the magnitude of the salt error of the indicator dyes (see Section IliA).

* M. Mvcobacterium_

Mouse

Rabbit

Jensen and Bainton [144]

Pater and Buyanovskaya [145]

Man

Neutrophils

Guinea-pig

Mandell [143]

Neutrophils

Mouse

Sprick [142]

Peritoneal macrophages

Peritoneal macrophages

Leucocytes

Leucocyte

Mouse

Rous [140,141]

Cell types

Animal

Investigator

Staphylococcus

avium 430

M. tuberculosis var.

hominis Ha7Rv

M. tuberculosis var.

Yeast

Candida

M. tuberculosis M. tuberculosis

M.* stregmatis var. hominis H37Rv M. tuberculosis var. hominis H37Rv

Agar

None

Matrix

Bromcresol green Bromphenol blue Bromcresol green Bromphenol blue Bromcresol green

Bromphenol blue

Bromphenol blue Bromcresol blue Bromcresol purple Neutral red

~ 5.2 ~ 4.6 ~ 5.2 ~ 4.6 ~ 5.2

~ 4.6

~ 4.6 4.0-5.5 ~ 5.0 ~ 6.5

~ 4.5 ~ 5.5 ~ 6.2 ~ 6.3 ~ 6.4 ~- 6.8 5.9-6.7 ~ 6.5 ~ 6.6 ~ 6.8

3.8-5.4 ~ 5.2 5,2 5.2-6.8

Bromcresol green Bromphenol blue Bromcresol green Bromcresol purple Bromphenol blue Bromcresol green Methyl red Litmus Chlorophenol red Bromcresol purple Bromthymol blue Brilliant yellow Neutral red Phenol red

j~ 4.6

Bromphenol blue

4.6

6 4 3 5

~

~ ~ ~ -~

pHi,

Bromphenol blue

Bromthymol blue Bromcresol green Bromphenol blue Litmus

Indicator

Leucocytes isolated from peritoneum after injection of dye and colour examined

Macrophages isolated 10 rain after injection of dye

Dye added to isolated leucocytes; medium pH 7.4

As above

Leucocytes isolated from peritoneum after injection of dye and colour examined

Remarks

INTRALYSOSOMAL pH ESTIMATES IN INTACT CELLS WITH MATRIX-BOUND OR FREE I N D I C A T O R DYES

TABLE I

4~ ta~

437 TABLE 1I 1NTRALYSOSOMAL pH CALCULATED FROM THE DISTRIBUTION OF A ~4CLABELLED WEAK BASE OR ACID ACROSS THE LYSOMAL MEMBRANE IN LYSOSOMES ISOLATED FROM RAT LIVER 'Tritosomes' were isolated from the livers of rats treated with Triton WR-1339 as described by Trouet [166]. 'Normal' lysosomes were isolated from the livers of untreated rats as described by Sawant et al. [64]; the purification factor was 20-25 it) comparison with the homogenate. Investigators

Type of lysosomes

T e m p e r - Composition ature (°C) of medium

Indicator

pHout pHln

Reijngoud and Tritosomes 25 coworkers [106,151l 25

130 mM KCI + MES/MOPS/Tris 250 mM mannitol + MES/MOPS/Tris

Methylamine Methylamine Methylamine Dimethylamine Trimethylamirte DMO

7.0 7.5 7.5 7.5 7.5 7.5

6.3 6.5 6.2 6.3 6.5 6.4

Reijngoud and Tager [153]

130 mM KC1 + MES]MOPS/Tris 250 mM marmitol + MES/MOPS/Tris

Methylamine

7.5

7.3

Methylamine

7.5

6.0

Tritosomes 0 0

Henning [155]

Tritosomes 0

350 mM sucrose + Tris-acetate

Methylamine

7.0 7.5

6.1 6.5

Goldmart and Rottertberg [154]

Normal

250 mM sucrose + HEPES

Methylamine

7.0

5.3

0

In Table II the published results obtained with the method based on the distribution of weak acids or bases are summarized. From the results obtained by Reijngoud et al. [106] it is clear that the calculated value is independent of the indicator compound used. The same value for the internal pH was obtained with three different bases (methylamine, dimethylamine and trimethylamine) and with the weak acid 5,5-dimethyl-2,4-oxazolidine dione (DMO). Two types of lysosomes were used in the studies described in Table II, lysosomes isolated from rats pretreated with Triton W R 1339, and lysosomes isolated from the livers of untreated rats. Essentially similar results were obtained with both types of lysosomes. Mego [167] and Reijngoud et al. [106] have attempted to estimate the intralysosomal pH by using the rate of degradation of radioactively labelled endocytosed protein as an indicator of the intralysosomal environment. Mego concluded from his experiments that the intralysosomal pH was 5, presumably at all pH levels tested. This conclusion was based on the observation that increasing concentrations of buffer of pH 5.0 had no influence on the degradation of endocytosed protein within intact lysosomes, in contrast to that at medium p H values of 4, 6, 7 and 8. According to Mego [167] the buffer ions tend to equalize the pH inside and outside the lysosomes. However, other explanations are possible. Reijngoud et al. [106] have assumed that the degradation of protein within

438 intact lysosomes responds to the pH in the lysosomal matrix in the same way it does when the degradation is catalysed by lysed lysosomes. They calculated that the pH inside the lysosomes was 5.0 and 6.0 at medium pH values of 7.0 and 7.5, respectively [140]. Recalculation of the data of Mego [167] yields a similar value (approx. 6.5) for the internal pH at a medium pH of 7.5 (c.f. ref. 167). Thus all three methods are in agreement in yielding values for the intralysosomal pH which are considerably lower than that of the ambient fluid, both in situ and in isolated lysosomes.

IIIC. The regulation of the intralysosomal p H The measurements summarized in Tables I and II confirm the hypothesis that the intralysosomal pH is low in comparison to that of the surrounding medium. This implies that a special mechanism must be present to bring about the pH difference across the lysosomal membrane. Two mechanisms are under current investigation, the first being that the low internal pH is brought about by an ATP-dependent proton pump, and the second that it is due to a Donnan type of equilibrium. 1. The ATP-dependent proton pump hypothesis. So far, the evidence for an ATP-driven proton translocation across the lysosomal membrane is only circumstantial. It is clear from the data summarized in Table II that a pH difference exists under conditions when it is improbable that an ATPase will be active, simply because of the absence of added ATP. This pH difference in isolated lysosomes (Table II) is comparable to the gradient existing across the lysosomal membrane in the intact cell (Table I). These observations alone make the presence and necessity of an ATP-driven proton pump equivocable, but the possibility cannot be excluded that an auxiliary mechanism may play a role in regulating the intralysosomal pH. Two main lines of investigation may be distinguished regarding the presence or absence of a membrane-bound ATPase in the lysosomes. The first concerns the effects of ATP on lysosomal processes and on the intralysosomal pH in isolated lysosomes, and the second embraces attempts to characterize a membrane-bound ATPase reaction. Mego and coworkers [168,169] have studied the effect of ATP on proteolysis in lysosomes isolated from mouse kidney and liver. They showed that ATP stimulated the intralysosomal degradation of endocytosed 12SI-labelled bovine serum albumin when the lysosomes were incubated at pH 8. At lower pH levels this effect was much less marked (see also ref. 103). Furthermore, Mego et al. [169] showed that GTP and ITP could replace ATP, although the effects of the former two compounds were less pronounced. Other nucleoside di- and triphosphates had no effect. Mg 2+ and Mn 2+ enhanced the stimulation by ATP, whereas Ca 2+ inhibited it. This stimulatory effect of ATP was also dependent on the buffer used; only a slight stimulatory effect on proteolysis was found if imidazole buffer was used instead of borate or bicarbonate, all three at a concentration of 25 mM [168]. Mego [170] also studied the effect of the uncoupler 2,4-dinitrophenol and the exchange-diffusion ionophore nigericin on the stimulation of proteolysis brought about by ATP. He

439 found that 2,4-dinitrophenol at a concentration higher than 0.2 m M could abolish the stimulatory effect of ATP. When nigericin was added to a medium containing 25 mM potassium borate (pH 8.0) there was a slight inhibitory effect on the proteolysis in the absence of ATP, whereas in the presence of ATP the stimulatory effect of the latter compound was diminished. According to Mego and coworkers [168] these observations can best be explained by the presence of an ATP-dependent proton-pumping system in the lysosomal membrane. This ATP-driven pump must be electroneutral. Mego [168] does not explicitly state whether the pump itself is electroneutral (i.e. that it brings about an exchange of H ÷ for K ÷ or Na ÷) or that it is electrogenic but coupled to a chargecompensating flux of Na ÷ or K ÷. The action of uncoupler and nigericin is explained by Mego [170] as a dissipation of the pH gradient. However, the results are difficult to interpret when one considers the mode of action of these compounds. It is not clear how an uncoupler, which brings about an electrogenic movement of protons, and nigericin, which brings about an electroneutral K+-H ÷ exchange, could bring about the same effect. The effects of ATP observed by Mego and coworkers [169] could be brought about by an ATP-induced increase of the stability of the lysosomal membrane. This would be detected as a stimulation of the intralysosomal hydrolysis of the labelled protein (see Section IIIB). Effects of ATP on the sedimentability of lysosomal enzymes or on the latency of these enzymes have been reported in the literature, but the underlying mechanism of action of ATP on the membrane stability is obscure [171-182]. Malbica [173,174] and Malbica and Hart [172], studying the effect of ATP on the sedimentability of acid phosphatase, fl-glucuronidase, arylsulphatase and hexosaminidase, found that in a crude lysosomal-mitochondrial fraction phosphate buffers containing ATP considerably decreased the leakage of acid phosphatase and fl-glucuronidase. In a purified lysosomal fraction, however, only the leakage of acid phosphatase was inhibited. A similar picture emerged when the influence of ATP was tested in the presence of compounds which bring about a labilization of the lysosomal membrane [173]; depending on the enzyme monitored and the type of preparation used, ATP was found to have either no effect or to abolish the destructive effect of the labilizer. A complicating factor in such studies is that the presence of compounds like Mg 2+, ATP and phosphate may promote adsorption of certain enzymes to membranes or membrane fragments. Huisman et al. [179,180] (see also ref. 178) tested the influence of ATP at low pH levels on the latency of cathepsin D, measured with haemoglobin, and cathepsin C, measured with an artificial substrate glycyl-phenyl-alanyl-p-nitroanilide. They found that whereas at p H 7.0 ATP stabilized the lysosomal membrane, at pH 4.5 it labilized the membrane, and at pH 5.0 it had no effect [179,180]. These results show clearly that the nature of the effect of ATP on the lysosomal membrane depends on the experimental conditions and on the parameters measured. Thus, no clear mechanism for the effects of ATP emerges. Mego et al. [169] reject the explanation that the stimulation of proteolysis

440 within the lysosomes on addition of ATP is a consequence of the stabilization of the membrane. This rejection is based on a single experiment in which the influence of ATP on the proteolysis of intralysosomal bovine serum albumin and on the sedimentability of the protein was studied. ATP was found to stimulate the proteolysis and to inhibit the release of intact protein from the lysosomes. Mego et al. [169] apparently corrected for the inhibition by ATP of the release ofsedimentable material in the following way. The amount of low molecular weight material produced by hydrolysis of protein in the intact lysosomes at any particular time was obtained by dividing the trichloroacetic acid-soluble radioactivity not by the total radioactivity but by the amount of sedimentable radioactivity that can be released by osmotic shock (cf. Figs. 8 and 9 of ref. 169). However, the validity of this procedure is doubtful, particularly when one realises that as the amount of sedimentable radioactivity decreases, the apparent hydrolysis will increase and approach infinity. As pointed out in Section IIIB, the two processes cannot be distinguished in this way. Furthermore, Mego et al. [169] assume that measurement of the sedimentability and of the increase in acid-soluble radioactivity yields two sets of data representing the two different processes, viz. the breakage of lysosomes and the hydrolysis of the protein (cf. ref. 146). Thus, the effect of ATP on intralysosomal proteolysis in isolated lysosomes may be due, not to the proposed effect on intralysosomal pH, but to other effects. In addition to the factors mentioned above, the impurity of the lysosomal fraction used must be taken into consideration. The fraction contains mitochondria and the effects of ATP could be due to mitochondrial ATPase activity, during which H ÷ is produced. Two groups of investigators [155,183] have studied the effect of ATP on the intralysosomal pH as calculated from the distribution of [~4C]methy]amine across the lysosomal membrane of 'tritosomes'; neither group has been able to detect any effect of ATP [155,183] even under conditions when according to other investigators [169,184,186] the pump should be active. Furthermore, ATP has no effect on the distribution of methylamine even in the presence o f a permeant anion like thiocyanate to provide a charge-compensating flux into the lysosomes together with the protons [183]. Two reports have appeared in which evidence for a membrane-bound ATPase activity in lysosomes has been brought forward. Iritani and Wells [184] have proposed that there is a HCO-3-stimulated Mg-ATPase in lysosomes. Their main argument is based on the isopycnic behaviour of this activity during centrifugation on a discontinuous sucrose density gradient of a mitochondrial-lysosomal fraction obtained from livers of Triton WR 1339-treated rats. However, the ATPase activity followed the distribution of cytochrome c oxidase closely, and not that of hexosaminidase, used as a marker for the lysosomes. This indicates that the ATPase activity was associated with the mitochondria rather than with the lysosomes. The association of a HCO-3-stimulated Mg-ATPase with the mitochondria has been reported by other investigators [185].

441 The suggestion by Hegner [186] that there is an ATPase specifically associated with the granules in leucocytes is based on inconclusive evidence; the activity described could be due, for instance, to a lysosomal pyrophosphatase. Kaulen et al. [187] tested the possibility that an ouabain-sensitive (Na + K)-ATPase (derived from the plasma membrane during formation of heterophagosomes) might be present in the lysosomal membrane. They were unable to detect any activity; the only ATPhydrolysing activity present in the lysosomal membrane fractions was sensitive to tartrate, an inhibitor of lysosomal acid phosphatase. Schneider [188] detected an ATPase activity in lysosomes isolated from the livers of Triton WR 1339-treated rats. Since this activity was latent and since the degree of latency was similar to that of acid phosphatase, Schneider [188] concluded that the ATP-hydrolysing activity was due to lysosomal acid phosphatases. However, as mentioned above and as shown by Brightwell and Tappell [189,190], ATP hydrolysis in lysosomes can also be brought about by acid pyrophosphatase. In conclusion, the evidence for a membrane-bound ATPase in the lysosomes that is able to pump protons into the lysosomes and thus regulates the intralysosomal pH is at present not convincing. 2. The Donnan type of equilibrium. Evidence for a Donnan equilibrium across the lysosomal membrane has been obtained from experiments with isolated lysosomes and 'tritosomes', in which the distribution of radioactively labelled weak bases like methylamine was used as a measure of the dpH. It has been found that at 0°C a classic Donnan equilibrium is set up in the lysosomal system, the membrane being permeable to small cations even in the absence of ionophores (refs. 153-155; see Section IIE). Goldman and Rottenberg [154] showed that the logarithm of the potassium gradient is numerically equal to the pH difference. From the data of Henning [155] it appears that the effectiveness of the alkali cations in decreasing the pH difference is as follows: Cs + ~ Rb + ~ K + Na ÷ > Li÷ >> Mg a÷ > Ca 2+. The relative ineffectiveness of Ca z+ and Mg z÷ may reflect their inability to permeate readily through the lysosomal membrane, even at 0°C, as can also be deduced from latency measurements. The rate of permeation of the alkali cations reported by Goldman and Rottenberg [154] and Reijngoud and Tager [153] differs considerably from that reported by Henning [155]. In the experiments of Reijngoud and Tager [153], for instance, equilibrium of both the K ÷ gradient and ApH occurred within 3 min, whereas in those of Henning [155] it took 60 min for equilibrium to be reached. Perhaps the difference in tonicity can explain the observed difference in entrance velocity; the high tonicity ( > 350 raM) employed in Henning's experiments may have acted as an impediment to a rapid flow of K ÷ into the lysosomes. At temperatures of 20 °C or higher, the permeability properties of the lysosomal membrane to small cations are completely different (see Section liE). At the higher temperatures the membrane is relatively impermeable to alkali cations and becomes readily permeable only in the presence of exchange-diffusion carriers [191] like nigericin, monensin and X 537 A [151,153]. Pore formers like gramicidin are also

442 INTRALYSOSOMAL pH AS A FUNCTION OF MEDIUM pH

7if I

I

I

I

5

6

7

8

g

PHout Fig. 3. Effect of medium pH on the intralysosomal pH in a KC1- and in a marmitol-containing medium. The vertical bars indicate the standard deviation (n ~ 5). For experimental details see ref. 106. • • , KC1; O O, mannitol. active in the lysosomal membrane, as shown by the fact that they bring about a decrease in ApH in the presence of NaC1 or KC1 [151]. In a sucrose or mannitol medium the intralysosomal pH increases from 4.5 at a medium pH of 5.0-7.6 at a medium pH of 8.5 (Fig. 3). A similar dependence of the intralysosomal pH on that of the medium is seen in a KCI medium at 25 or 37°C in the absence of ionophores (Fig. 3). This response of the intralysosomal pH to that of the medium indicates that acid-base equivalents must permeate across the lysosomal membrane. The magnitude of the response indicates that the transport of acid-base equivalents must be electroneutral (see Section IliA). In agreement with this postulate is the observation that a transport of K ÷ can be induced by exchange diffusion carriers like nigericin [151,153]. A similar transport can be induced by a combination of the electrogenic ionophore valinomycin and a protonophore (Fig. 4)*.

IIID. Conclusion To explain the data discussed in Section III the following mechanism is proposed in which a Donnan-type distribution of acid-base equivalents across the lysosomal membrane plays an essential role. The lysosomal matrix is an isoionic protein solution [192-194] containing glycoproteins with low isoelectric points as the main constituents. An isoionic protein solution is defined as a solution that contains, apart from dissolved protein, no ions other than those arising from dissociation of the solvent, e.g. O H - and H + in the case of water. The pH of an isoionic protein solution can be shown to be close to the isoelectric point if only one protein is present, or to the weighted average of the isoelectric points if different proteins are present in solution. The glycoproteins in the lysosomes have, in general, a low * Our initial failure to induce a K+-H+ exchange in lysosomeswith valinomycinplus uncoupler [151] was due to the use of concentrations of these compounds too low to be effective.

443 EFFECT OF VALINOMYCIN AND ON INTRALYSOSOMAL pH l

l

7.5

UNCOUPLER i

[Vatin°mycin1~Iml)"

6.5~

~ I

0

10

20 [ccP] ~M

I

30

z.o

Fig. 4. Effect of valinomycin and uncoupler on intralysosomal pH. Lysosomes, isolated from livers of rats pretreated with Triton WR-1339 [106] were incubated in a medium (final volume 1 ml), containing 130 mM KCI, 10 mM MES, 10 mM MOPS, sufficient Tris to give a final pH of 7.5, 1 ~ ethanol, valinomycin and CCP at the concentrations indicated, 3H20 and [~*C]methylamine. In parallel experiments l~*C]sucrose was present instead of [~4C]methylamine. After incubation at 25°C for 1 rain, the distribution of [~4Clmethylamine was determined as described in ref. 106. pHln was calculated as described in ref. 106.

isoelectric point [195], thus in an isoionic solution this will lead to a low intralysosomal pH. In addition, acidic groups (including acidic glycolipids [62]) on the inner side of the lysosomal membrane m a y also play a role in bringing about a low p H [196,62]. The response of the intralysosomal pH to the extralysosomal conditions is then solely determined by the properties of the lysosomal membrane, which at 20 °C or higher is relatively impermeable to small cations but permeable to acid-base equivalents. At these temperatures, exchange-diffusion ionophores have an effect on the ApH in the presence of the appropriate cations. At 0°C the membrane is readily permeable not only to acid-base equivalents but also to small cations. This results in a classic Donnan equilibrium. In this respect the experiments of Saifer and Steigman [115] are illustrative in showing that a Donnan distribution for protons and sodium is set up across the membrane of a dialysis bag containing an isoionic albumin solution. In this system, ApH and the logarithm of the Na ÷ gradient are numerically equal. In model systems, the presence of impermeant anions inside a vesicle can lead to an acidification of the interior of the vesicle, provided that an electroneutral movement of protons across the membrane can take place. Thus Blok et al. [197] have shown that when valinomycin plus uncoupler are added to liposomes containing the K ÷ salt of an impermeant anion, acidification of the interior of the vesicle Occurs.

One difficulty remains. In experiments with isolated lysosomes, a slow permeation of K + can sometimes be observed, particularly after prolonged incubation. (Casey, R., unpublished observations; see also ref. 153). The question arises of whether a similar slow permeation occurs in vivo. If this were so, a Donnan equilibrium would eventually be set up leading to an increase in intralysosomal pH. To

444 counteract this increase in pH, protons would have to be pumped into the lysosomes in exchange for K +. For the reasons discussed in Section IIID, an energy-dependent pump would have to bring about a non-electrogenic proton flux (cf. the ATP-driven, vectorial K+-H + exchange in gastric mucosa [198,199]). However, it is possible that the lysosomal membrane is not permeable to K + under physiological conditions and that the slow permeation of K + across the lysosomal membrane observed in isolated lysosomes is due to changes in the lysosomal membrane occurring during incubation in vitro.

IV. GENERAL CONCLUSIONS The lysosomes are regarded as the main site of intracellular degradation of macromolecules, the degradation being catalysed by hydrolases with an acid pH optimum. During hydrolysis acid-base equivalents and low-molecular weight compounds are produced. This review has dealt with the mechanism of diffusion of these products out of the lysosomes. It is at present impossible to specify what the mechanism of diffusion is. Research in this area is still in its infancy. In this respect, it is important to realize that the composition of the lysosomal matrix can easily be altered, since extracellular material can be taken up by the process of endocytosis. It should thus be possible to bring about well-defined alterations in the lysosomal matrix by allowing the endocytosis to take place of carefully chosen, specially designed macromolecules like Dextran sulphate or Dextran DEAE. By introducing such indiffusible anions or cations into the lysosomal matrix, it should be possible to change the ApH across the lysosomal membrane. Furthermore, it should be possible to fill the lysosomes with indicator dyes covalently bound to an indigestible matrix and subsequently to monitor changes in the ApH in the isolated organelles. It should also be possible to label specifically lysosomes from different cell types by making use of the phenomenon of 'homing' of macromolecules (specific endocytosis by a certain cell type only) [200]. By this procedure it should in principle be possible to see whether or not differences exist between the membrane properties of lysosomes from different cell types in the same tissue. These approaches, making use of the phenomenon of endocytosis, should greatly facilitate the study of the permeability properties of the lysosomal membrane. Finally, it would be of great interest to have an endogenous probe of intralysosomal conditions. One possibility is to use the spectral properties of myeloperoxidase, which is present in the granulae of polymorphonuclear leucocytes, for this purpose. Since the spectral properties of isolated myeloperoxidase are dependent on the pH and the ionic composition of the medium, it might be possible to monitor the permeation of acid-base equivalents and of ions across the lysosomal membrane in isolated granulae spectrophotometrically, which would be of particular value for kinetic studies (suggestion of Dr. R. Wever).

445 ACKNOWLEDGEMENTS The a u t h o r s are very grateful to Karel v a n D a m , Machiel Blok, Bob Casey a n d M a r i a H o l l e m a n s for helpful discussions a n d for their critical c o m m e n t s o n the manuscript.

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