The Biochemistry of Manganese

Symposium on Trace Elements

The Biochemistry of Manganese Merton F. Utter, Ph.D. *

Manganese is widely distributed in nature but occurs only in trace amounts in biological materials, particularly in animal tissues. Even where the concentrations are highest as in bone, liver, or lactating mammary gland, manganese is present only in amounts of 2 to 3 parts per million, by weight. In spite of these very small amounts, it is clear that manganese plays at least one, and probably several, important roles in the maintenance of life and biologic functions. The earliest studies to demonstrate this point were nutritional experiments which started about 1930 by McCollum, Elvehjem, Hart, and others. The attempts to elucidate the biologic function of manganese can be divided into three different categories. In the first, the nutritional approach just mentioned, the effects of removing or depleting manganese in the diet are observed in terms of growth, reproduction, survival, and other in vivo and in vitro functions of experimental animals. In the second, which I have chosen to call biochemical, the distribution of manganese within tissues or within cells may be investigated. Perhaps more importantly, in this approach, attempts are made to define the effects of manganese on biochemical processes, such as the role of the metal ion in enzymatic reactions. In the third approach, which is not possible with all trace elements but which has been fruitful in the case of manganese, the effects of excess metal ion can be investigated. This last approach will be considered elsewhere in this symposium. A major problem in determining the biological role(s) of manganese has been to correlate the information derived from the three approaches just noted. As we will see, there is ample evidence to indicate that manganese deficiency produces a number of striking effects in the experimental animal. Similarly, manganese has been implicated in a number of metabolic and enzymatic processes. It has not been particularly easy to explain the effects of manganese deficiency in the whole animal in *Professor and Chairman, Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio Supported in part by Grant AM 12245 of the National Institutes of Health and by Atomic Energy Contract AT-(1l-1)-1242.

Medical Clinics of North America- Vol. 60, No. 4, July 1976

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terms of specific in vitro observations but there are now some plausible partial answers to these questions and more complete answers should be forthcoming. Effects of Manganese Deficiency on Experimental Animals Let us consider first some of the effects observed in experimental animals during manganese deficiency. Table 1 summarizes some of a very large number of observations of this type. First, the lack of sufficient manganese results in some very dramatic general effects such as a lowered rate of growth, failure to reproduce, and shortened life span. Although these observations are obviously of fundamental importance, they do not lend themselves easily to explanations at the biochemical level. The second group of disorders related to manganese deficiency shown in Table 1 has been more useful in this regard. As indicated, certain postural and skeletal defects have been noted in experimental animals. In the chicken, the most striking effect is perosis or slipped tendon. In the rat, manganese deficiency before birth results in a failure of the otoliths to develop properly in the inner ear with subsequent problems related to equilibrium. This phenomenon is discussed in considerable detail elsewhere in this symposium. The failure of the tendons and the otoliths to develop normally in manganese deficiency seems to be related to more extensive connective tissue abnormalities, probably involving the failure of collagen and mucopolysaccharide formation. This has given rise to one of the most promising areas of investigation of the biological role of manganese and we will return to these studies shortly. It should be noted, however, that there are other probable abnormalities in manganese deficiency as shown in the third part of Table 1. These include neurologic symptoms, structural abnormalities and impaired function in mitochondria, impaired blood clotting, and possible alterations in glucose metabolism. It should be emphasized that this list could be lengthened considerably. Manganese Ion as an Activator of Enzymes Several hypotheses can be advanced to explain the effects of manganese deficiency in terms of biochemical mechanisms. One of the most likely explanations is the participation of manganese in enzymatic reactions in one or more metabolic areas. Although this idea will be explored further, other roles for manganese can be postulated. For example, manganese might be an essential element in some structural material. This function cannot be excluded, particularly since the metal is present in very small amounts, and it becomes technically difficult to study. It is also possible that manganese may play some special role as a biological ion. It is concentrated in the mitochondria, for example, and a transport or similar function may exist for the ion such as has been shown in other circumstances for calcium ion. It seems more likely, however, that manganese participates mainly in the action of the enzymatic machinery, and an examination of possible roles will constitute the main substance of the remainder of this discussion. There are two major ways in which manganese may participate

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Table 1. Effects of Manganese Deficiency on Experimental Animals Impaired rate of growth, failure to reproduce, shortened life span!'·2! Postural, skeletal defects Porosis (slipped tendon)!5 Ataxia (inner ear).· 28 Other (all seem to be related to deficiency in mucopolysaccharide formation in connective tissue) Other suggestions Neurological symptoms' Impaired mitochondrial oxidation!' Impaired blood clotting' Abnormal glucose metabolism (pancreas)'

in enzymatic reactions. Manganese may act as a dissociable cofactor which is required for the activity of the enzyme. This may involve chelation of the metal ion with a phosphate-containing substrate, an in teraction of the free metal ion with the enzyme or still other types of interaction. However, in all of these modes of participation the metal ion is free to dissociate and is not a permanent part of the enzymatic structure. In the second type of manganese participation in enzymatic reactions, the metal ion is held firmly by the protein to form a metalloprotein and the metal ion plays an important role in the function or the structure of the enzyme. These two types of possible functions of manganese in enzymatic reactions will be discussed separately, starting with cases in which the metal acts as a dissociable cofactor. Literally hundreds of different enzymes may be stimulated by addition of manganese under in vitro conditions. The types of enzymes involved (Table 2) include kinases, thioesterases, peptidases, deacylases, decarboxylases, and certain enzymes involved in biosynthetic processes, such as the glycosyl transferases. In addition, there is adenyl cyclase, the enzyme involved in the formation of cyclic AMP. This list is by no means a complete one. There are two difficulties here. First, the list is too inclusive and it seems unlikely that all or even most of these classes of enzymes will be shown to be specifically involved in the effects of manganese deficiency. Second, in most and perhaps all of these cases, the effects are not specific for manganese. That is, manganese is not the only metal ion that will stimulate these reactions. Many times magnesium also serves as an activator and often, as an apparently more effective one. In other cases, metal ions such as nickel, cobalt, iron, and zinc can also act as activators. The problem becomes one of relating the results of in vitro experiments to in vivo effects, such as those seen in manganese deficiency. In spite of these difficulties, there is by now a fairly compelling body of evidence which suggests that manganese may have a special relationship to the biosynthetic processes which lead to the formation of the polysaccharide moieties of mucopolysaccharides and related compounds. It will be recalled that this area has been previously linked with some of the overt symptoms of manganese deficiency (Table 1).

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Table 2.

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Partial List of Types of Enzymes that can be Stimulated by MN2+

ENZYME

SUBSTRATE(S)

Phosphatases Kinases Thioesterases Peptidases (some) Dehydrogenases (some) Decarboxylases Glycosyltransferases Adenyl cyclase

Phosphate esters ATP, etc. Thioesters Peptides, proteins Various Carboxylic acids UDP galactose, etc. ATP

Possible Role of Manganese in Mucopolysaccharide Biosynthesis Figure 1 illustrates a summary of one of the more attractive hypotheses for the role of manganese, in this case, in the biosynthesis of chondroitin sulfate, one of the mucopolysaccharides. The vertical structure on the left represents the polypeptide backbone of chondroitin sulfate to which is attached a carbohydrate side chain which has three sugar moieties, xylose and two molecules of galactose, followed by the main carbohydrate portion of this molecule, a repeating unit of glucuronate and N -acetyl galactosamine. A sulfate moiety is also attached to each disaccharide of this part of the structure. It has been noted by Leach and colleagues 14 • 16 and by others22 that manganese is a far more efficient divalent cation than magnesium in stimulating the incorporation of the various carbohydrates into this structure. The components may be put on in a stepwise fashion moving from left to right in Figure 1 and from the step in which xylose is added outward, the reactions appear to proceed better if manganese is present at the points indicated in Figure 1. If this is so - that manganese is required for the formation of chondroitin sulfate and probably other similar substances - then it is possible to see why manganese deficiency may cause problems in the connective tissue. It might be useful to examine some typical data dealing with the effect of manganese on mucopolysaccharide synthesis. Figure 2 was adapted from some experiments performed a few years ago by Robinson, Telser and Dorfman22 on the incorporation of 14C galactose into

Mn2+ O-Xylose -

~

Mn2+

Mn2+

Mn2+

l

l

~

Gal -

I

Gal- [Glucuron -

r-

50 4

N-Acetylgal]n Mn 2,+

Figure 1. Postulated role for manganese in chondroitin-sulfate synthesis (mucopolysaccharides). (Adapted from Leach, R. M., Jr.: Fed. Proc., 30:991-994,1971, with permission.)

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1000 •

z

ILl I-

0 et:

Q..

(!)

~

......

...... ~

•

800

Mn2+

~.

600 400

:E Q.. u 200

/.-.--.

Mg2+

•

10 20 30 40 50

[Mg2+]or [Mn 2+] (mM) Preparation from Chick Embryo Epiphyses. Figure 2. Relative effects of Mn2+ and Mg2+ on incorporation of "C-galactose into chondroitin sulfate-protein. (Adapted from Robinson, H. C., Telser, A., and Dorfman, A.: Proc. Nat. Acad. Sci. (V.S.), 56:1859-1866, 1966, with permission.)

chondroitin sulfate protein in a chick embryo extract. This experiment illustrates several typical points concerning this type of experimentation. First, it has often been necessary to use tracer techniques and relatively crude acceptor systems to demonstrate mucopolysaccharide synthesis. Second, in many of these systems, manganese is dramatically more effective than magnesium as shown by the differences in the two curves in Figure 2. Finally, at higher concentrations manganese often becomes inhibitory. So it would appear from a number of experiments of this sort that manganese is very effective in promoting the incorporation of carbohydrates into mucopolysaccharides. Unfortunately, the situation may not be quite that simple. If we look at the position of manganese in the periodic table, we find this element is located in the middle of the first long series (see Table 3). It is in this series that we find the so-called transition elements and manganese is surrounded by a number of closely related elements including chromium, cobalt, copper, and so on. Therefore, it is not only magnesium that one must worry about in terms of the activation of enzymatic reactions by divalent metal ions. A number of these other ions must be considered. For example, when Leach et aP6 examined the relative effectiveness of different metal ions on two reactions of mucopolysaccharide synthesis, polysaccharide synthetase and galactose transTable 3. Manganese and the Transition Metals A ------------ v

Cr

18

24

23

Ii\ful ~

Fe

Co

Ni

Cu

Zn----------- Kr

26

27

28

29

30

36

From the first long series of the periodic table. The numbers refer to the atomic numbers of the elements.

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Table 4. Comparison of Effectiveness of Different Metal Ions on Polysaccharide Polymerase and Galactose Transferase Activity':' GALACTOSYL METAL ION

POLYMERASE

TRANSFERASE

(10 mM) Mg2+ Mn 2 + Co2+ NP+ Fe 2 +, Zn 2 +, Cu 2 +

0.08 1.00 0.75 0.33 0

0.06 1.00 1.33 0.44 0

*Data from Leach, R. M., Jr., Muenster, A. M., and Wien, E. M.: Arch. Biochem. Biophys., 133 :22-28, 1969, with permission.

ferase, they found (Table 4) that manganese indeed was much more effective than magnesium in activating these two enzymes. However, if other ions were examined it was noted that cobalt and nickel were also about as effective as manganese. Thus, we have to worry not only about the comparisons with magnesium, but also the relative effects with a number of other metal ions. One point should be added here with reference to the results shown in Table 4. Leach and his colleagues, after seeing the effect of cobalt here, tried feeding cobalt to manganesedeficient animals. This did not relieve the symptoms. This series of experiments illustrates very well the complexities and difficulties of interpreting in vitro experiments in terms of in vivo effects. A number of factors must be considered in any extrapolation from the in vitro results. The first matter is one of specificity of the metal effect. Many studies are not complete enough to permit a judgment of the specificity question. As noted in Figure 2, manganese was inhibitory if tested at concentrations that were too high, so a wide range of concentrations must be tried. Second, different metal ions may have different interactions with the substrates of a reaction. Accordingly, a kinetic . study with different metal ions may have to include variations in the substrate concentrations also. Third, the effects of the presence of other ions on any particular metal ion should be known. It is apparent that a large number of parameters may have to be investigated and that the number of experiments required may be prohibitive. Another set of factors is of crucial importance in the evaluation of hypotheses arising from in vitro experiments with metal ions. This concerns the need for knowledge of the effective concentration of the metal ion in the tissue. The concentration of a metal ion in a biological material is influenced by a number of factors including transport, intracellular distribution, and particularly, the binding of the metal ions to other substances such as phosphate esters. For example, cobalt binds considerably more tightly to ATP than does magnesium. Manganese is somewhere in between. The amount of "free" or unbound cobalt or other metal ion will therefore be dependent on the concentrations of other substances that may be present. As a further factor to be considered, there have been recent reports2 that the presence of polyamines, such as spermine, markedly lowers the concentration of manganese required for

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a galactosyltransferase involved in mucin biosynthesis. In spite of these considerable difficulties, the utilization of information on the biochemical functions of manganese as obtained by in vitro experiments can be useful, particularly if combined with in vivo experiments to test new hypotheses as they arise. The probable involvement of manganese in mucopolysaccharide biosynthesis is a good example of the process of using in vivo information to buttress in vitro findings and vice versa. Manganese as a Component of Metalloproteins I would like to turn now to a somewhat different subject-that of the metalloproteins containing manganese. In contrast to the enzymes where manganese acts as a dissociable cofactor as discussed above, manganese is built rather firmly into the protein in these examples. Table 5 shows a list of manganoproteins compiled according to the information available to me. The first protein on the list was reported a number of years ago to be present in serum and to be involved in the transport of manganese. 29 Only two manganoenzymes have been reported. The first is pyruvate carboxylase which we first found in avian liver but which appears to be present in liver and kidney of all species as well as in certain other sources. The avian liver pyruvate carboxylase is ordinarily a manganoprotein26 but this is not necessarily so for some other varieties of the enzyme. The second enzyme is superoxide dismutase8 also found in avian liver as well as in certain bacteria. These are the only well-established examples of manganoenzymes known at this time. There are three other manganoproteins whose function are not entirely understood. The first is so-called avimanganin23 which was found in avian liver. As we will see later, this protein may be related to superoxide dismutase. Two other proteins containing manganese have been reported in plants - concanavalin A from jack bean! and manganin4 from peanuts. The exact function of the metal ions in these proteins is not clear but they may be necessary structural components. It is probably appropriate to say a few words about the occurrence and metabolic functions of the two reported manganoenzymes, pyruvate carboxylase and superoxide dismutase. Pyruvate carboxylase was found a number of years ag030 and has been the subject of a number of studies in our laboratory and by others. Figure 3 summarizes some of the information concerning this enzyme including the reaction catalyzed by the enzyme, its distribution, and particularly, its metabolic function,3! In liver, kidney, and other gluconeogenic tissues, the enzyme appears to Table 5. Metalloproteins Containing Manganese PROTEIN

Mn-transferrin Pyruvate carboxylase Superoxide dismutase Avimanganin Concanavalin A Manganin

FUNCTION

Transport Enzyme Enzyme Enzyme (?) Structural (?) Structural (?)

SOURCE

Serum Avian liver, many other sources Avian liver, bacteria Avian liver Jack bean Peanut

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Reaction Pyruvate + HC0 3

+

MgATP

F.

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Me 2 + K+ ' ) Oxalacetate + MgADP + Pi

Occurrence Liver, kidney, brain, adipose tissue, many microorganisms Metabolic Function Lactate, Alanine

~

IPyruvate 4

Oxalacetate

+

I4

Phosphopyruvate 4

Glucose, Glycogen

Dicarboxylic Amino Acids Figure 3.

Occurrence and function of pyruvate carboxylase.

catalyze the first step of carbohydrate synthesis from pyruvate. The pathway leads from pyruvate to oxalacetate, then to phosphoenolpyruvate and after a series of steps to glucose. The reaction may have another function in other tissues such as brain where it may help resupply oxalacetate that has been diverted for other purposes such as the formation of dicarboxylic amino acids. In animal cells the enzyme is a large (molecular weight of 500,000) biotin-containing enzyme with four subunits. 32 In avian liver, the metal ion is normally manganese.26 In some other species such as calf liver the metal ion appears to be a mixture of manganese and magnesium. 24 In yeast, where the enzyme is about the same size and is generally similar to the liver enzyme, the metal ion is zinc. 27 Thus, the enzyme is a manganoenzyme only in certain cases. This fact becomes important for the arguments concerned with the possible role of this enzyme in the effects of manganese deficiency. The function of manganese in the pyruvate carboxylase is reasonably well understood. One of the interesting characteristics of manganese, which can be touched on only briefly here, is that the metal ion provides an unusual probe for studies of enzymatic structure and mechanism. This is true because manganese is paramagnetic, and the presence of the unpaired electron endows the metal ion with certain magnetic properties that are very useful in nuclear magnetic or electron spin resonance studies. In these ways manganese has provided an effective tool for elucidating the interactions of dissociable manganese, substrate, and enzyme. Cohn and her colleagues have been especially active in such studies 29 and much of what we know about the biochemistry of manganese at the mechanistic level comes from this sort of work. However, in many cases, the question remains unanswered as to whether manganese is the naturally occurring metal for these enzymes or is simply a useful biochemical probe. In addition to these studies in which manganese is present as a dissociable metal ion, manganese has been useful where it occurs in the metalloenzymes. Here, it serves as a reference point for structural studies of the active site. This is particularly true for pyruvate carboxylase. Figure 4 shows the current view of the spatial relationship of the bound manganese in avian liver pyruvate carboxylase and one of the substrates, pyruvate. From the earliest days

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after the discovery of manganese in this enzyme, the metal ion has been considered to have some special relationship to pyruvate. 20 Later studies by Fung and Mildvan9 and by Scrutton, Reed, and Mildvan25 have produced the picture shown in Figure 4 where the distances between manganese and different portions of the pyruvate molecule can be estimated. This involves a triangulation procedure where the spatial relationships of manganese and other paramagnetic atoms such as 13C have been measured. Studies of this type appear to be unusually promising for enzymes where manganese is naturally present or can be introduced. Other studies will also give information concerning the role of the metal ion in the reaction. For example, it has been suggested that manganese in pyruvate carboxylase "activates" the methyl group of pyruvate by virtue of the electron withdrawing properties of the metal ion. The methyl group of pyruvate serves as the acceptor for carbon dioxide in the formation of oxalacetate. Unfortunately, although these studies are of considerable inherent interest, they may not help us a great deal in trying to decide why manganese is necessary for life and certain biological functions. That is, the problem remains one of distinguishing between nonessential and essential functions for the metal ion. The second known manganoenzyme is superoxide dismutase. Figure 5 shows the nature of the substrate for this enzyme, the superoxide radical, the reaction catalyzed by the enzyme, and an example of a possible biological source of the superoxide radical. As shown in Figure 5, superoxide is formed by the partial reduction of oxygen, that is, reduction by a single electron rather than by two electrons, in which case the peroxide ion would be formed. Fridovich and his colleaguesB have carried out extensive studies on this enzyme. They suggest that the superoxide radical or some substance formed readily from it may be deleterious to biological materials. Although the superoxide radical can be dissipated by non-enzymatic reactions, superoxidedismutase, a very commonly distributed enzyme, tends to remove it by a dismutation reac-

1+---8.5 A-----+I

o / O=C \1+--7.1 A-----+I H-C

/

C=O

I'H

H

I(

7.4A--~ PYRUVATE

Figure 4. Relationship of bound Mn in pyruvate carboxylase to pyruvate. (Adapted from Fung, C. H., and Mildvan, A. S.: Biochemistry, 12:620-629,1973, with permission.)

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SUPEROXIDE

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DISMUTASE

Substrate

® ® 2- 2H 2 + 02 --'--~) 02 --~) 02 ) H2 0 2 Oxygen Superoxide Peroxide Hydrogen Peroxide Radical Ion

IEnzyme I Reaction 2H+

+

02 + 02 Superoxide ) 02 + H20 2 . Dismutase

Biological Source of Superoxide Flavin Substrates + 02 Oxidases Figure 5.

Oxidized ) Substrates

+ H20 2 + 02

Superoxide dismutase.

tion in which one molecule of the superoxide is oxidized to molecular oxygen while a second is reduced to peroxide. The formation of superoxide is believed to occur through the partial reduction of oxygen by various flavin oxidase. Normally, these enzymes form hydrogen peroxide, but if in some cases the reduction was only half completed, superoxide radical would result. Fridovich and his colleagues have found several types of superoxide dismutase. One of the most common varieties contains copper and zinc. These proteins have been known for many years 17 and were often called cupreins but their enzymatic function had not been suspected. This type of superoxide dismutase is found in erythrocytes, or in the cytosolic fraction of many types of animal cells}2,18 A little later, it was found that E. coli,.. StreptococcU8,33 and most recently chicken liver mitochondria34 contain a manganoform of superoxide dismutase. There is still a third iron-containing variety of the enzyme, also found in E. coli. 35 Possible Roles of Pyruvate Carboxylase and Superoxide Dismutase in Manganese Deficiency One of the main interests in this discussion is whether one or both of these enzymes, pyruvate carboxylase or superoxide dismutase, plays any important role in the effects of manganese deficiency. This is a difficult question23 but some partial answers may now be suggested. Before dealing with this question, it will be useful to describe one other aspect of the manganoprotein story, that involving avimanganin which Scrutton26 isolated from chicken liver mitochondria. When studying pyruvate carboxylase in chicken liver as a manganoprotein, we 23 fed chickens radioactive manganese and this provided a handy tool for studying the stability of the metal ion in the enzyme. It was found to be very firmly bound and could be removed only by extensive denaturation of the enzyme. During these studies, it was also noted that there was a second 54Mn-containing protein in chicken liver mitochondria. Several years later, Scrutton isolated this other fraction and purified it to apparent

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AVIMANGANIN Chicken liver mitochondria (only 2 Mn - proteins)

~ DEAE Sephadex Pyruvate carboxylase

IAvimanganinl Mol. wt. 89,000 Inactive as superoxide dismutase

Figure 6.

Avimanganin.

homogeneity and called it avimanganin. Figure 6 shows the essential facts concerning avimanganin as described by Scrutton. The protein has a molecular weight of about 89,000 and appears to be the only other manganese-containing protein in avian liver mitochondrial extracts. One of the final purification steps in the purification procedure involved chromatography with DEAE Sephadex, an important point in the discussion to follow. By the time avimanganin was isolated, Scrutton was aware of the report of a mangano-superoxidismutase in E. coli. He tested avimanganin for such activity but unfortunately could not detect any evidence for this reaction. In spite of this failure to demonstrate superoxide dismutase activity in avimanganin, Table 6 shows a number of interesting similarities in the two proteins. They are found in the same part of chicken liver mitochondria, the matrix. There seem to be similar amounts of the two protein present; the molecular weights are similar; and both proteins contain manganese (111).8.23 Furthermore, it will be recalled that chicken liver Initochondria appeared to contain only one other mangano-protein in addition to pyruvate carboxylase. Therefore, it seemed reasonable to suggest that avimanganin was superoxide dismutase except for the disturbing fact that avimanganin had no enzymatic activity. Weisiger and Fridovich34 have provided a plausible explanation for this apparent discrepancy. They passed purified superoxide dismuTable 6.

Comparison of Avimanganin and Mn-Superoxide Dismutase*

Same source Chicken liver mitochondria Same location Mitochondrial matrix Similar amounts of protein reported Similar molecular weight (80,000 vs 90,000) Both contain Mn (Ill) Superoxide dismutase 90% inactivated by DEAE Sephadex ':'Data from Scrutton, M. C.: Biochemistry, 21 :3897, 1971, and Weisger, R. A., and Fridovich, I.: J. BioI. Chem., 248:3582,1973.

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tase over DEAE Sephadex to mimic the final step of the purification of avimanganin. The superoxide dismutase was about 90 per cent inactivated by this treatment. Therefore, it is reasonable to postulate that avimanganin may represent an inactivated form of superoxide dismutase. If we can assume that avimanganin is really superoxide dismutase, it lends additional interest to some experiments Scrutton carried out a few years ago23 to find out whether manganese deficiency led to any abnormalities in pyruvate carboxylase and avimanganin. He fed chickens on three diets, one very low in manganese with only 0.3 mg of manganese per kg, a second with a normal amount of manganese present, and a third diet with a low but significant amount of the metal present (Table 7). The chickens grown on the very deficient diet showed the typical signs of manganese deficiency such as perosis while those on the intermediate diet showed some symptoms. Those on the high manganese diet were normal. Pyruvate carboxylase was then isolated from the livers of chickens grown on the three types of diets. Table 7 shows that the amount of manganese in pyruvate carboxylase from chickens on the very low manganese diet contained only 0.07 nanomoles per mg of protein as compared with 6.1 in the enzyme from the normal animal. The intermediate diet gave values in between. Although not shown here, other studies indicated that manganese in the enzymes from chickens fed manganese-poor diets has been replaced by magnesium. 24 Thus, there is approximately an amount of magnesium in the low-manganese enzyme equivalent to the missing manganese. The activity of pyruvate carboxylase appeared to be essentially the same whether or not the enzyme contained manganese or magnesium. This is the most important point of these studies as far as the present discussion is concerned. This was disappointing in that the results appear to show that manganese plays only a nonessential role in this enzyme and therefore that the essential or specific function of manganese must lie elsewhere. The avimanganin part of these experiments may be more fruitful. As shown in Table 7, in livers obtained from chickens grown on the deficient diet, there is a very striking decrease in the amount of manga-

Table 7. Effect of Manganese Deficiency on Pyruvate Carboxylase and Avimanganin in Chicken-Liver Mitochondria" CONC. MN IN

DIET

(mg/kg)

4.8

0.3

58

Condition of chickens

Perosis

Some symptoms

Normal

Pyruvate Carboxylase Mn/mg protein'''' Amount (activity)

0.07 Normal

1.4 Normal

6.1 Normal

Avimanganin Mn/mg protein" * Amount (protein)

3.1 Very low

16.5

16.9 Normal

'Data from Scrutton, M. C.: Biochemistry, 21 :3897, 1971. **Nanomoles/mg protein.

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725

nese in the avimanganin fraction (3.1 as compared with 16.9 nanomoles per mg of protein). Since avimanganin is not enzymatically active, the amounts of this protein could not be estimated by measurements of this type. Rather, chromatographic analyses were carried out to try to determine the amount of avimanganin present. These tests showed a very low amount in this fraction in the case of the manganese-deficient diet. This result suggests either that avimanganin is not present in the absence of manganese or that it has a different chromatographic behavior. If avimanganin is not formed in the absence of manganese or fails to survive under these circumstances and if avimanganin is really superoxide dismutase, we may have an enzymatic factor linked to manganese deficiency. It must be added that a, direct demonstration of diminished superoxide dismutase activity during manganese deficiency would appear to be an important next step in testing this hypothesis. If some relationship between manganese deficiency and superoxide dismutase can be demonstrated, the question still remains as to the biological consequences of this situation. FridovichB believes that the superoxide radical or something closely related to it can cause widespread damage to a variety of biological materials including membranes. As noted earlier, mitochondrial damage and impaired function have been well established as one of the effects of manganese deficiency.

Concluding Remarks The present status of the biochemical role(s) of manganese may be summarized as follows. First, it is clear that manganese plays one or more essential roles in the maintenance of life and biological functions. Second, it seems likely, although by no means certain, that manganese fulfills this role(s) in conjunction with the action of enzymes. Third, where manganese is acting as a dissociable cofactor of an enzyme, it is difficult to correlate and evaluate the wealth of information already available from in vitro studies on the effects of manganese on various isolated enzyme systems. It is particularly difficult in these cases to establish that manganese plays a specific role since in most or all cases, metal ions other than manganese can also fulfill the role of the dissociable cofactor. Fourth, only a handful of proteins have been reported where manganese is incorporated firmly into the protein to form a metalloprotein. Only two of this small number of manganoproteins have been shown to be enzymes, pyruvate carboxylase and superoxide dismutase. Even with these two examples, the available evidence suggests that manganese may play a specific role for superoxide dismutase but not for pyruvate carboxylase. Fifth, in the various metabolic areas in which manganese might play an important role, the reactions leading to the biosynthesis of mucopolysaccharides have been strongly implicated. The evidence is based on the presence of apparent defects in connective tissues during manganese deficiency and on the marked ability of manganese to activate a number of enzymes involved in the biosynthesis of this class of compounds. The glycosyltransferases are a prime example. There seems to be no compelling reason to believe that manganese will play a specific and essential role in a single metabolic area such as in the biosynthesis of mucopolysaccharides. Two other areas appear to

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be promising areas for further investigation. The first of these concerns the mangano-form of superoxide dismutase which has been found in avian liver but which probably has a much wider distribution. If this is true and this enzyme can be shown to have significant protective effects against a harmful biological substance, some of the effects of manganese deficiency may be shown to be connected with this enzyme. A second area for investigation might involve the glycoproteins. Glycosyltransferases are active in the synthesis of these compounds in a wide variety of situations. Glycoproteins are now believed to play crucial roles in many biological functions including the action of hormones, blood clotting, lactation, membranes, and so on. The foregoing list was selected because there have been reports in each case of some impairment during manganese deficiency.

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