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Lactate dehydrogenases in poikilotherms: Definition of a complex isozyme system
Comp. Biochem. Physiol., 1966, Vol. 18, pp.
261 to 269. Pergamon Press Ltd. Printed in Great Britain
LACTATE DEHYDROGENASES IN POIKILOTHERMS: D E F I N I T I O N OF A COMPLEX ISOZYME SYSTEM P. W. HOCHACHKA Ramsay-Wright Zoological Laboratories, University of Toronto, Toronto 5, Canada, and Department of Biochemistry, Duke University, Durham, North Carolina (Received 9 November 1965) Abstract--1. Five kinds of subunits (A-E) are involved in generating 14 isozymes of lactic dehydrogenase (LDH) in skeletal muscle of Salmo namaycush and S. fontinalis. One group of 5 LDHs, migrating near the origin at neutral
pH, is derived from random assembly of D and E subunits into tetramers. Another essentially independent series of 9 LDHs, migrating anodaUy at neutral pH, are formed by a non-random assembly of subunits A, B and C to yield A4, A3B1, A2B2, A1Bs, B4, BsC1, BaC~, B1Cs and C4. 2. Liver of lake trout contains 5 LDHs homologous to the A-B series in muscle, but only the homopolymer B4 is abundant. Brook trout liver has the B-C series with only C4 in abundance. Liver of S. gairdneri has only the A4 homologue. 3. The LDHs formed in dissociation-reassociation tests and in interspecies crosses are consistent with the 5-subunit model advanced. INTRODUCTION THE epaxial muscle of 2 closely related Salmonidae, S a l m o fontinalis (brook trout) and S . namaycush (lake trout) commonly contains 14 electrophoretically resolvable lactic dehydrogenase (LDH) isozymes. A group of 9 L D H forms (numbered 1-9 from the anodal end of zymograms) migrates rapidly anodally at pH 7; a second group of 5 LDHs (10-14) scatters on anodal and cathodal sides of the origin (Fig. 1, row M). Such an occurrence of more than 5 LDHs, although not unusual in cold blooded vertebrates (Hochachka, 1965; Markert & Faulhaber, 1965), cannot be accounted for by the 2-subunit model adopted for mammalian LDHs. An integral part of the 2-subunit model assumes the random assembly of 2 kinds of subunit polypeptidic chains in all possible tetramer combinations to yield 5 compositionally distinct proteins (see Markert, 1963b). Most previous studies observing more than 5 LDHs have assumed that a third subunit is present and can aggregate with either A or B to form new kinds of L D H tetramers. Indeed, the evidence for polypeptidic variants of A or B (see for example, Kraus & Neely, 1964) in higher vertebrates and for a third subunit, C, in testes (Zinkham et al., 1964) is unequivocal. Guided by these results, accounts of more than 5 L D H s in lower vertebrates also have assumed a third subunit, C, which on assorting randomly with A and B could yield 15 isozymes (Goldberg, 1965 ; Adams & Finnegan, 196S). 261
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Such assumptions, based largely or solely on numbers of resolvable L D H bands, probably are inadequate. Instead, it appears that at least 5 subunits must be involved in generating the large n u m b e r of L D H isozymes occurring in tissues of the lower vertebrates. Three subunits, A, B and C, are involved in generating 9 anodally migrating L D H s . In vivo, A and B polymerize usually preferentially to form the 5 most electronegative L D H ' s , A 4, AaB1, A2Be, A1B3, and B~. Subunits B and C can assemble to form 4 more components, B3C1, B2C2, B1C.~ and C 4. A and C normally do not interact, but individuals apparently lacking in B subunits show that A and C can "hybridize" to form another and different system of 5 L D H s , A4, AaC1, ..., to C 4. Subunits D and E polymerize quite independently of A, B and C, and form 5 L D H s (D4, D:~E1, ..., to E4) which at p H 7 characteristically migrate near the origin. These D and E subunits do not "hybridize" with A, B or C to any appreciable extent; similarly, if A, B and C subunits assembled into tetramers by a strictly random mechanism, 15 rather than 9 L D H s would be produced. T h e results thus indicate that assembly of L D H s in this example is not a fully random process. T h e evidence for, and some predictions of, a 5-subunit model are examined in this paper. MATERIALS AND METHODS
Experimental animals All of the trout used in these experiments were yearlings, 4-5 in. in length, and about 50-100 g in weight. The animals were adapted (for several months) to 6-7°C and a lightdark cycle of 12 hr. They were fed a commercial fish food once every 2 days. Through the courtesy of Dr. F. E. J. Fry of the University of Toronto, it was possible to sample a population of Splake trout, interspecies hybrids obtained by crossing lake trout females with brook trout males. Only a small number of lake and brook trout individuals contributed to the F1 pool of Splake.
Preparation of LDH Samples of tissues were excised and homogenized at ice temperature in distilled water, phosphate-citrate buffer (ionic strength, 0"052, pH 7), 0"1 M tris HC1 buffer, pH 8, or 0"88 M sucrose. Similar LDH patterns were obtained using any of these extraction media. Whenever the LDH preparation would be extensively concentrated (by drying under vacuum at 0-5°C), distilled water was used for homogenizing. Usually a wet weight of about 1 g was used. In the cases of heart, brain and liver, the entire tissue was excised and homogenized. The homogenate was spun in a Servall refrigerated centrifuge (3-5°C, SS-34 head) at about 28,000 g for 60 min. The supernatant was spun for another 120 min and used as a source of LDH activity. This high speed supernatant yielded LDH isozyme patterns identical to preparations which were filtered through a 50 cm Sephadex (G-100) column.
Gel electrophoresis A Buchler type electrophoresis unit was used. Starch gel (Connaught Laboratories, University of Toronto, Canada) was prepared as described by Smithies (1955). Electrode vessels held a phosphate-citrate buffer (ionic strength, 0"52, pH 7) and were bridged with four thicknesses of Whatrnan No. 1 filter paper. Depending upon L D H activity in any given sample, 5-10 lambdas were placed onto 3 mm square wicks of filter paper. The wieks were then inserted into slits cut into the starch gel and established quite a sharp origin. Usually, an amount of LDH activity was placed on the gel equivalent to a decrease
MODEL FOR A COMPLEX L D H S Y S T I ~
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in absorbance of 0.40-0.80 per rain at 3 x 10 -4 M reduced nicotinamide adenine dinucleotide ( N A D H ) and 3 x 10 -3 M pyruvate (Hochachka, 1965). LDH activity in gels was visualized by described staining techniques (see, for example, Markert, 1963a). Control incubations, with substrate absent, indicated one cathodally migrating band at pH 7. This kind of component has been observed previously (see Shaw, 1964; Markert & Faulhaber, 1965). It is not shown in any of the zymograms in this paper.
Sephadex gel filtration Sephadex G-100 was prepared as previously described (Hochachka, 1965). Dissociation-reassociation experiments Crude LDH preparations were taken up in 1 M NaCI (Markert, 1963b) containing 0"2 M phosphate (Chilson et al., 1965) at pH 8-5 and were frozen overnight at -25°C. Freeze rates were kept slow to promote efficient dissociation (Chilson et al., 1965). Experimental tests consisted of mixing LDH samples from rainbow trout liver (A4 or LDH-1), lake trout liver (B4 or LDH-5) and brook trout liver (Ca or LDH-9) in all combinations taken two at a time. Control tests contained LDHs from only one liver source, this being largely A4, B4 or C4. After slow thawing, the preparations were dialyzed against distilled H20 for 12 hr at 4°C to remove NaC1 and allow subunit reassociation. The resulting LDHs were resolved by gel electrophoresis in the usual manner. RESULTS AND DISCUSSION
The muscle LDHs A detailed examination of Fig. 1 (rows M and m) illustrates the close similarity between muscle L D H s of lake and brook trout. For purposes of clarity the proposed subunit formulae of the lake trout series are given in small letters; the subunit formulae for brook trout muscle L D H s are written in capital letters. T h e A - B ( L D H 1-5) series in both species show quite similar, mobilities towards the anode. T h e lake trout 1-5 series shows activity usually skewed in favor of a4 or L D H - 1 . In the brook trout A-B series, the L D H activity is distributed binomially between the L D H s 1-5. All the components between L D H s 5-9 can be resolved in brook trout; in lake trout, it is unusual to find by our methods L D H s , 6 7 or 8 (b3cl, b2c~ and blcz), though L D H - 9 (ca) is clearly detectable. Always a certain amount of L D H activity migrates between L D H s 9 and 10; this is variable within and between species, for reasons which will be discussed below. L D H s 10-14 ( D - E series) in brook and lake trout have nearly identical electrophoretic mobilities. In view of these close similarities and the phylogenetic relationship between the two species, it is clear that each muscle L D H band in one is homologous to its counterpart in the other species. Spacings between the L D H s 10-14 indicate that these differ from each other by equal increments of charge. This is likewise true for the A - B and B - C series. However, the spacings between L D H s 10-14 are larger by a factor of 1.5 than those between, say, the L D H s 1-5. This result indicates independent components making up One complex isozyme system and such interpretation is further supported by the L D H patterns in liver tissue. Liver LDHs and the A, B and C subunits. Unlike muscle, liver in both species displays only 1 major L D H component, with 4 minor forms (usually undetectable
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by gel-staining methods) separating out at equal distances from each other. The number and charge properties of the liver L D H s fit the assumption of classical 2 subunit systems (Markert, 1963b), one subunit being produced very much in excess of the other; assembly is presumably random and yields 5 tetramers. As is evident in Fig. 1 (row 1 and L), however, the liver L D H s in lake trout are not the direct homologs of the liver L D H s in brook trout, a most striking observation in view of the close similarity of the muscle LDHs. Brook trout liver possesses L D H s 5-9, L D H - 9 (or C4) being in very high concentration, L D H s 5-8 appearing in lesser and lesser activity. In lake trout liver, the L D H 1-5 series is preponderant, L D H - 5 (or b4) having the highest activity, LDH-1 (or aA) having an activity so low it is also often undetectable by the gel-staining method (see Fig. 1, row L). Simultaneous 24 hr electrophoresis of liver and muscle L D H s from both brook and lake trout indicates that the main brook trout liver L D H and muscle CA (LDH-9) are indeed the same proteins, that the main lake trout liver L D H is identical to muscle LDH-5 or b 4 and so forth. That is to say, the liver and muscle systems are in fact homologous to each other and thus our use of similar nomenclature is probably justifiable. Tissue specificity derives from the relative abundance of different subunits, and, hence, different L D H proteins rather than from tissue-specific isozymes p e r se (see Markert, 1963a, for further discussion of this point). It is accepted that electrophoretic criterion of identity of different L D H s is not unequivocal. However, it is consistent with the data of Nissebaum et al. (1964) showing that mammalian L D H s of identical electrophoretie mobilities but of different tissues are apparently identical proteins. Presumably, then, each tissue in Salmonidae has the potentiality of synthesizing all of the subunits, though only in muscle is this potentiality expressed. Our interpretation of the trout liver L D H s predicts that L D H - 9 (C~, brook trout liver) and L D H - 5 (b4, lake trout liver) be homotetramers. Two kinds of evidence support the prediction: 1. Female lake trout x male brook trout crosses (called Splake) have been made and are available in abundance (Fry & Gibson, 1953). Now if the major liver L D H s are indeed "pure" tetramers (C Ain brook trout, b Ain lake trout), one outcome of the above cross would be the generation of 3 new kinds of LDHs, C3bl, C2b2 and Clb3, to yield 5 L D H s (Shaw, 1964). This result indeed is obtained (Fig. 2). Further details on interspecies inheritance will be published elsewhere. For the moment, it is gratuitous to our argument that when these 5 L D H s occur in Splake liver (middle row, Fig. 2), they occur in roughly a 1 : 4 : 6 : 4 : 1 ratio, that predicted by assuming a random assembly mechanism. This kind of distribution of L D H activity in the liver isozymes is not seen in either of the parental species (about 70 specimens of each species tested). 2. Since assembly of tetramers is thought to be epigenetic (Markert, 1963a), it should be and indeed is possible to generate the C3b ~, C~b 2 and Cab 3 isozymes in vitro. New LDHs, analogous to those seen in Splake liver, can be obtained by dissociation and reassociation of brook trout liver L D H and lake trout liver LDH.
BROOK
123456789 %
to C4
84
11
04
12
13
14 E4
FIG. 1. Resolution of trout LDHs on starch gel. Electrophoresis conditions: 18 hr at 35-40 mA and 200 V, in phosphate-citrate buffer, pH 7, ionic strength 0.52. Gel temperatures constant at lo-12°C. Activity per unit wet weight is approximately proportional to intensity of staining. Anode at left of all electrophoretograms. Origin marked by dark dashes. (1) Lake trout liver LDH. (m) Lake trout epaxial muscle LDH. (L) Brook trout liver LDH. (M) Brook trout muscle LDH. Subunit formulae for brook trout LDHs in capital letters; for lake trout LDHs, in small letters.
LIVER
CDH 64
oripn
FIG. 2. Liver LDHs of lake and brook trout and of one result of an F, hybrid between the two species (called Splake). Electrophoretic conditions as in Fig. 1. Time of run 18 hr. The liver LDH patterns for the hybrid trout (Splake) are a Out of about 70 brook and lake trout unique consequence of hybridization. examined, Splake kind of liver LDH patterns were never found.
A4
%Cl
*2=2
Al%
C4
origin I
ACC
1
A
A+
*4
64
=4
I
B
t
B+c
FI
origin
FIG. 3. Dissociation-reassembly experiments with A,, II, and C1 LDHs. Conditions of dissociation and reassociation given in text. Electrophoresis conditions as in Fig. 1. Time of electrophoresis, 15 hr. Subunit formulae given on the electrophoretogram.
FIG, 4. Comparison of LDH patterns in high speed supernatants of lake trout muscle homogenized in 0.88 M sucrose and distilled water. Electrophoresis condiI$ hr. LDHs 1-5 and LDH 9 (6, 7 tions as in Fig. 1. Time of el ec t rophoresis, and 8 not usually present in lake trout muscle) show a drop in activity in sucrose homogenates. Presumably, this drop in activity is due to removal during centrifugation of osmotically shrunk nuclei and/or mitochondria. LDHs lo-14 show Experiments with heart LDHs yielded similar essentially no change in activity. results.
MODEL FOR A COMPLEX L D H SYSTEM
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Fig. 3 (row B + C) shows that in one freeze-thaw cycle, dissociation and reassembly is incomplete but the C3bl, C2b~, Ctb 3 forms predicted by the model are clearly demonstrable. The generation of 3 new LDHs, in vivo by an interspecies cross, and in vitro by dissociation and reassociation of lake and brook trout liver LDHs, establishes that the main liver isozyme in each species is made up of one kind of subunit, and that 4 subunits aggregate to make up the enzyme protein. That is, C4 (LDH-9) and b 4 (LDH-5) are homotetramers (see Shaw, 1964, for discussion of limitations of this approach). On the assumption that tissue-specificity resides in isozyme expression and not in isozyme kind, we now can define unequivocally the 2 homologous isozymes in muscle L D H zymograms. The pertinent observation is that both C 4 (LDH-9) and B 4 (LDH-5) are within the muscle zymogram series (see Fig. 1, row M). A 3-subunit model based on random assembly yielding 15 LDHs allows only 1 "pure" tetramer, say B4, within the series; the other 2 "pure" tetramers must be terminal in zymograms, say anodally, A4, and cathodally, C 4. These experiments thus rule out the 3-subunit model (Goldberg, 1965; Markert & Faulhaber, 1965) as an explanation of multiple LDHs in lower vertebrates. Adams & Finnegan (1965) found that the presence of terminal bands tended to be variable, appearing at some stages in development but not at others. We have seen an analogous case in brain tissue LDHs of warm- and cold-adapted goldfish (Hochachka, 1965). It was therefore important to have evidence that the band which we label LDH-1 (A4) in this paper is indeed a homotetramer made of only A subunits. Three lines of evidence justify accepting LDH-1 as a "pure" tetramer: 1. If LDH-1 is made up of 1 subunit kind, subunit interactions with B would yield L D H patterns consistent with the bandings for brook and lake trout muscle and for lake trout liver (Fig. 1). 2. A-C interactions, when occurring, ought to likewise generate 3 new kinds of LDHs. In brook trout muscle and heart tissue, the gene for subunit B is frequently completely turned off, no B subunits being formed. In these cases, A and C subunits form the expected 5 LDHs, Aa, A3C 1.... , C a (Hochachka, unpublished). 3. If the assumption of a homotetramer is correct, dissociation-reassociation of LDH-1 should not, and does not, yield any new kinds of LDHs (Fig. 3, row A). To verify this we took advantage of S. gairdneri (rainbow trout) liver which possesses only a single L D H form, electrophoretically similar to brook trout LDH-1. Freeze-thaw treatment of homogenates with only LDH-1 activity does not generate new L D H forms on removal of salt (Fig. 3, row A). On the other hand, A-B and A-C interactions can be shown under identical circumstances to produce the predicted heteropolymers. Thus, mixtures of A4 and Ba yield on reassociation the expected 3 new forms, A3B1, A~B~ and A1B 3 (Fig. 3, row A+B). Similarly, mixtures of A4 and C4 produce 3 new forms, A3C1, AaC ~ and A1C 3 (Fig. 3, row A+C). These tests thus suggest a validity to the definition of LDH-1 as the homotetramer, A 4.
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P . W . Hocnncx-mn
Parenthetically, it is perhaps worth stressing that Fig. 3 represents results from one freeze-thaw cycle. It is evident that B-C hybridization is not complete but that A-B and A-C polymerizations have essentially gone to completion. This result is consistent with the L D H patterns in epaxial muscle, which show a greater abundance of A-B hybrids than of the B-C kind, but it does not account for the usual in vivo absence of A-C tetramers. The D - E system in trout muscle
If we accept the subunit model for LDHs, the fastest cathodal L D H within a series is presumably a "pure" tetramer; LDH-14 is thus assigned arbitrarily the subunit formula E 4. Anodal to LDH-14 (Fig. 1, row M) are 4 components separated by equal distances, but, as mentioned above, the interband spacings here are larger than in the L D H 1-9 group. (The disparity in spacings is clearer after 24 hr electrophoresis.) This suggests that the D - E series form an isozyme system largely independent of the A - B - C series and predicts that LDH-10 is a homopolymer to which we can assign the subunit formula D 4. Some amphibian L D H systems seem to include a set of components of this kind (Wright, 1965). Further work will be required to define these L D H s with greater confidence. In most zymograms, L D H activity between L D H - 9 and LDH-10 (between C 4 and D4) cannot be well resolved. This activity is not an artifact of the gel-staining procedure and is possibly formed by a certain amount of hybridization between the A - B - C group and the D and E subunits. Indeed, this sort of intermediate activity might be predicted by our model. Lack of resolution of the L D H between C 4 and D 4 and its variability between individuals presumably is related to the degree to which the mechanisms for non-random subunit assembly are not failsafe. That the assembly mechanism cannot be a random one is indicated by the development of 9 rather than 15 L D H s within the A - B - C complex. Were the A, B and C subunits to associate into tetramers by a random process, tissues like trout epaxial muscle would possess at least 20 LDHs, 15 in the A - B - C series plus the 5 in the D - E group. Reports approaching this number of isozymes have in fact been made (see Markert & Faulhaber, 1965; Wright, 1965) and may represent greater degrees of freedom in the assembly of L D H subunits than is evident in the trout tissues under consideration. Nature of constraints on subunit aggregation Akhough this area is largely unexplored, there are indications of at least two mechanisms potentially capable of restricting subunit assembly. One constraint appears to be related to cellular compartmentalization. Evidence from fractionation studies is difficult to obtain for LDH, which is typically very water-soluble (see, for example, Novikoff, 1960). In the usual kind of homogenates used in this study, intracellular organelles are osmotically swelled or disrupted. To overcome this problem, samples from a given muscle tissue were divided into equal wet weights and homogenates were prepared in either 0"88 M sucrose or distilled water. The
MODEL FOR A COMPLEX LDH SYSTEM
267
usual procedure was then used in preparing the homogenates for gel electrophoresis. Centrifugation time and speeds were sufficiently high to spin down nuclei and mitochondria, but no attempt was made to fractionate the latter. L D H patterns of only high speed supernatants were assayed. After centrifugation of the sucrose homogenates (at about 30,000 g), there was a very noticeable drop in L D H s 1-5, a lesser disappearance of LDH-9, but essentially no change in the L D H s 10-14 when these were compared to L D H patterns from water supernatants (Fig. 4). The effect is not due to a specific sucrose inhibition for the L D H assay was performed after some 18 hr of electrophoresis in a gel medium which is free of sucrose. When similar tests were performed using lake trout heart, a highly aerobic tissue which possesses only an abundance of the A - B - C groups of L D H (Hochachka, unpublished), sucrose preparations yielded a very noticeable drop in L D H activity in all the isozymes to about the same extent. These preliminary experiments raise the probability of a cytoplasmic L D H system (the D - E group) separated from nuclear and/or mitochondrial ones (the A - B - C group), a situation possibly similar to that for malate dehydrogenases (Thorne et al., 1963). The presence in trout heart and brain of only A - B - C combinations (Hochachka, unpublished) might supply biological reasons for suspecting mitochondrial rather than nuclear associated activity. Since both nuclear and mitochondrial L D H activities are known to occur (see, for example, Novikoff, 1960), these preliminary tests do not allow a distinction to be made in the case of trout muscle. Tentatively, however, it appears that cellular compartmentalization may allow one means, a biological one, for restricting random subunit assortment. A second constraint relates to molecular size of the different LDHs. A few preliminary runs suggest that the fast anodal A-B tetramers (LDHs 1-5), assumed to be of the same molecular weight as other vertebrate LDHs, 140,000 g/mole (Fine et al., 1963; Daugherty, 1965), filter more rapidly than the D - E series through G-100 Sephadex and can be readily separated from the D - E L D H s in one run on a 50 cm column. The D - E tetramers filter at rates identical to hemoglobin and hence are tentatively assumed to be about one-half the molecular weight of the A-B LDHs. L D H - 9 (C4) seems to be intermediate in size. Appella (in Kaplan, 1964) has noted that the mammalian L D H "subunit" consists of two smaller components, each about 18,000 mol. wt. It is an interesting conjecture that the D - E series in trout muscle are tetramers formed from these smaller subunits. This entire question in the case of trout muscle L D H s requires much further work before confidence can be placed in these first experiments. They do, however, raise the possibility that size differences between the A-B and the D - E subunits affect monomer aggregation. This sort of mechanism, combined with other restrictions such as cellular compartmentalization, assures that assortment of the 5 subunits in trout muscle is quite unique rather than totally random. Dissociationreassociation tests show that also within the A - B - C group certain polymerizations seem to occur more readily than others. To what extent this might be due to size differences or to other more subtle structural restrictions is likewise a question which must be left open for future investigation.
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SUMMARY 1. By taking advantage of tissue and species homologies, a model involving 5 kinds of subunits is proposed to account for the multiple L D H s commonly found in lake and brook trout and perhaps other lower vertebrates. T h r e e subunits, A, B and C, generate a group of 9 anodally moving L D H s . L D H s 1-5 (the A - B group) can be written A4, AsB1, A2B2, A1B 3 and B 4. B and C polymerizations yield tetramer L D H s 6-9 (B3C 1, B2C2, B1C s and Ca). A - C interactions normally do not occur in vivo, but the expected A - C tetramers can be generated readily in dissociation-reassembly experiments. Similarly, the A-B and B-C heterotetramers can be formed in vitro. T h e dissociation-reassembly studies show that random assembly of the A, B and C subunits can yield 15 kinds of isozymes. T h e presence in brook and lake trout of only 9 forms implies that subunit assembly is at least partially directed. In cases where fewer constraints on subunit assembly occur, all 15 of the possible A - B - C tetramers would presumably be expressed. 2. D and E subunits are postulated to account for L D H s 10-14 (D4, D3E 1, D~E2, D1E 3 and E4), a group of 5 L D H s migrating near the origin at neutral pH. Since interband spacings, molecular weights and cellular location are probably different from L D H s 1-9, it is assumed that these form an essentially independent isozyme system. T h e r e is no evidence for D or E subunits interacting with the A, B or C monomers. 3. Probably all tissues are potentially capable of producing all 5 subunits, but only in muscle is this potentiality expressed in the species under study. Acknowledgements--Some facilities for this research were made possible through a grant from the National Research Council (Canada) to Dr. K. C. Fisher, University of Toronto. The development of the problem owes much to discussion with Geneva H. Williams. Part of the study was carried out while working as a postdoctoral fellow with Dr. W. L. Byrne, Department of Biochemistry, Duke University (U.S.P.H.S. Grant No. GM-06628-06S1).
REFERENCES ADAMS E. & FINNEGANC. V. (1965) An investigation of lactate dehydrogenase activity in early amphibian development. `7. exp. Zool. 158, 241-251. CHILSON O. P., COSTELLO L. A. • KAPLAN N. O. (1965) Studies on the mechanism of hybridization of lactic dehydrogenases in vitro. Biochemistry 4, 271-281. DAUGHERTY W. (1965) Analysis of the apparently single form of lactate dehydrogenase found in the flatfish, Paralichthys denatatus. Am. Zoologist 5, 205. (Abstract only.) FINE I. H., KAPLANN. O. & KUFrlNECD. (1963) Developmental changes of mammalian lactic dehydrogenases. Biochemistry 2, 116-121. FRY F. E. J. & GIBSONM. B. (1953) Lethal temperature experiments with speckled trout × lake trout hybrids..7. Heredity 44, 56-57. GOLDBERGE. (1965) Lactate dehygrodenases in trout: evidence for a third subunit. Science, N.Y. 148, 391-392. HOCHACHKAP. W. (1965) Isoenzymes in metabolic adaptation of a poikilotherm: Subunit relationships in lactic dehydrogenases of goldfish. Archs. Biochem. Biophys. 111, 96-103. KAPLAN N. O. (1964) Lactate dehydrogenase--Structure and function. In Subunit Structure of Proteins. Biochemical and Genetic Aspects, Brookhaven Symposia in Biology, No. 17, 131-153.
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KRAUSA. P. & NEELY C. L., JR. (1964) Human erythrocyte lactate dehydrogenase. Four genetically determined variants. Sdence, N . Y . 145, 595-597. MARKET C. L. (1963a) Epigenetic control of specific protein synthesis in differentiating cells. In Cytodifferentiation and Macromolecular Synthesis, 21st Symp. Soc. Study Dev. Growth (Edited by LOCKE, M.), pp. 64-84. Academic Press, New York. MARrmRT C. L, (1936b) Lactic dehydrogenase isozymes : dissociation and recombination of subunits. Science, N . Y . 140, 1329-1330. MAmC~T C. L. & FAULHAB~RI. (1965) Lactate dehydrogenase isozyme patterns of fish. jY. exp. Zool. 195, 319-332. NISSPBAVMJ. S., PACKERD. E. & BODANSHX"O. (1964) Comparison of the actions of human brain, liver and heart lactic dehydrogenase variants on nucleotide analogues and on substrate analogues in the absence and in the presence of oxalate and axamate..7, biol. Chem. 239, 2830-2834. NOVIHOFFA. B. (1960) Electron transport enzymes: biochemical and tetrazolium staining studies. In Histochemistry and Cytochemistry, Proc. 1st Int. Congr. 1960 (Edited by WEGMAN, R.), pp. 465-481. Macmillan, New York. SHAW C. R. (1964) The use of genetic variation in the analysis of isozyme structure. In Subunit Structure of Proteins. Biochemical and Genetic Aspects, Brookhaven Symposia in Biology, No. 17, 117-130. SMITHIES O. (1955) Zone electrophoresis in starch gels: group variations in the serum proteins of normal adults. Biochem. 3. 61, 629-641. THORNE C. J. R., GROSSMANL. I. & KAPLANN. O. (1963) Starch-gel electrophoresis of malate dehydrogenase. Biochem. biophys. Acta 73, 193-203. WRIGHT D. A. (1965) The development of lactate dehydrogenase isozymes in intraspecific crosses in the genus Rana. Am. Zoologist 5, 205. (Abstract only.) ZINKHAMW. H., BLANCOA. & KUPCYHKL. (1964) Lactate dehydrogenase in pigeon testes: genetic control by three loci. Science, N . Y . 144, 1353-1354.
261 to 269. Pergamon Press Ltd. Printed in Great Britain
LACTATE DEHYDROGENASES IN POIKILOTHERMS: D E F I N I T I O N OF A COMPLEX ISOZYME SYSTEM P. W. HOCHACHKA Ramsay-Wright Zoological Laboratories, University of Toronto, Toronto 5, Canada, and Department of Biochemistry, Duke University, Durham, North Carolina (Received 9 November 1965) Abstract--1. Five kinds of subunits (A-E) are involved in generating 14 isozymes of lactic dehydrogenase (LDH) in skeletal muscle of Salmo namaycush and S. fontinalis. One group of 5 LDHs, migrating near the origin at neutral
pH, is derived from random assembly of D and E subunits into tetramers. Another essentially independent series of 9 LDHs, migrating anodaUy at neutral pH, are formed by a non-random assembly of subunits A, B and C to yield A4, A3B1, A2B2, A1Bs, B4, BsC1, BaC~, B1Cs and C4. 2. Liver of lake trout contains 5 LDHs homologous to the A-B series in muscle, but only the homopolymer B4 is abundant. Brook trout liver has the B-C series with only C4 in abundance. Liver of S. gairdneri has only the A4 homologue. 3. The LDHs formed in dissociation-reassociation tests and in interspecies crosses are consistent with the 5-subunit model advanced. INTRODUCTION THE epaxial muscle of 2 closely related Salmonidae, S a l m o fontinalis (brook trout) and S . namaycush (lake trout) commonly contains 14 electrophoretically resolvable lactic dehydrogenase (LDH) isozymes. A group of 9 L D H forms (numbered 1-9 from the anodal end of zymograms) migrates rapidly anodally at pH 7; a second group of 5 LDHs (10-14) scatters on anodal and cathodal sides of the origin (Fig. 1, row M). Such an occurrence of more than 5 LDHs, although not unusual in cold blooded vertebrates (Hochachka, 1965; Markert & Faulhaber, 1965), cannot be accounted for by the 2-subunit model adopted for mammalian LDHs. An integral part of the 2-subunit model assumes the random assembly of 2 kinds of subunit polypeptidic chains in all possible tetramer combinations to yield 5 compositionally distinct proteins (see Markert, 1963b). Most previous studies observing more than 5 LDHs have assumed that a third subunit is present and can aggregate with either A or B to form new kinds of L D H tetramers. Indeed, the evidence for polypeptidic variants of A or B (see for example, Kraus & Neely, 1964) in higher vertebrates and for a third subunit, C, in testes (Zinkham et al., 1964) is unequivocal. Guided by these results, accounts of more than 5 L D H s in lower vertebrates also have assumed a third subunit, C, which on assorting randomly with A and B could yield 15 isozymes (Goldberg, 1965 ; Adams & Finnegan, 196S). 261
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Such assumptions, based largely or solely on numbers of resolvable L D H bands, probably are inadequate. Instead, it appears that at least 5 subunits must be involved in generating the large n u m b e r of L D H isozymes occurring in tissues of the lower vertebrates. Three subunits, A, B and C, are involved in generating 9 anodally migrating L D H s . In vivo, A and B polymerize usually preferentially to form the 5 most electronegative L D H ' s , A 4, AaB1, A2Be, A1B3, and B~. Subunits B and C can assemble to form 4 more components, B3C1, B2C2, B1C.~ and C 4. A and C normally do not interact, but individuals apparently lacking in B subunits show that A and C can "hybridize" to form another and different system of 5 L D H s , A4, AaC1, ..., to C 4. Subunits D and E polymerize quite independently of A, B and C, and form 5 L D H s (D4, D:~E1, ..., to E4) which at p H 7 characteristically migrate near the origin. These D and E subunits do not "hybridize" with A, B or C to any appreciable extent; similarly, if A, B and C subunits assembled into tetramers by a strictly random mechanism, 15 rather than 9 L D H s would be produced. T h e results thus indicate that assembly of L D H s in this example is not a fully random process. T h e evidence for, and some predictions of, a 5-subunit model are examined in this paper. MATERIALS AND METHODS
Experimental animals All of the trout used in these experiments were yearlings, 4-5 in. in length, and about 50-100 g in weight. The animals were adapted (for several months) to 6-7°C and a lightdark cycle of 12 hr. They were fed a commercial fish food once every 2 days. Through the courtesy of Dr. F. E. J. Fry of the University of Toronto, it was possible to sample a population of Splake trout, interspecies hybrids obtained by crossing lake trout females with brook trout males. Only a small number of lake and brook trout individuals contributed to the F1 pool of Splake.
Preparation of LDH Samples of tissues were excised and homogenized at ice temperature in distilled water, phosphate-citrate buffer (ionic strength, 0"052, pH 7), 0"1 M tris HC1 buffer, pH 8, or 0"88 M sucrose. Similar LDH patterns were obtained using any of these extraction media. Whenever the LDH preparation would be extensively concentrated (by drying under vacuum at 0-5°C), distilled water was used for homogenizing. Usually a wet weight of about 1 g was used. In the cases of heart, brain and liver, the entire tissue was excised and homogenized. The homogenate was spun in a Servall refrigerated centrifuge (3-5°C, SS-34 head) at about 28,000 g for 60 min. The supernatant was spun for another 120 min and used as a source of LDH activity. This high speed supernatant yielded LDH isozyme patterns identical to preparations which were filtered through a 50 cm Sephadex (G-100) column.
Gel electrophoresis A Buchler type electrophoresis unit was used. Starch gel (Connaught Laboratories, University of Toronto, Canada) was prepared as described by Smithies (1955). Electrode vessels held a phosphate-citrate buffer (ionic strength, 0"52, pH 7) and were bridged with four thicknesses of Whatrnan No. 1 filter paper. Depending upon L D H activity in any given sample, 5-10 lambdas were placed onto 3 mm square wicks of filter paper. The wieks were then inserted into slits cut into the starch gel and established quite a sharp origin. Usually, an amount of LDH activity was placed on the gel equivalent to a decrease
MODEL FOR A COMPLEX L D H S Y S T I ~
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in absorbance of 0.40-0.80 per rain at 3 x 10 -4 M reduced nicotinamide adenine dinucleotide ( N A D H ) and 3 x 10 -3 M pyruvate (Hochachka, 1965). LDH activity in gels was visualized by described staining techniques (see, for example, Markert, 1963a). Control incubations, with substrate absent, indicated one cathodally migrating band at pH 7. This kind of component has been observed previously (see Shaw, 1964; Markert & Faulhaber, 1965). It is not shown in any of the zymograms in this paper.
Sephadex gel filtration Sephadex G-100 was prepared as previously described (Hochachka, 1965). Dissociation-reassociation experiments Crude LDH preparations were taken up in 1 M NaCI (Markert, 1963b) containing 0"2 M phosphate (Chilson et al., 1965) at pH 8-5 and were frozen overnight at -25°C. Freeze rates were kept slow to promote efficient dissociation (Chilson et al., 1965). Experimental tests consisted of mixing LDH samples from rainbow trout liver (A4 or LDH-1), lake trout liver (B4 or LDH-5) and brook trout liver (Ca or LDH-9) in all combinations taken two at a time. Control tests contained LDHs from only one liver source, this being largely A4, B4 or C4. After slow thawing, the preparations were dialyzed against distilled H20 for 12 hr at 4°C to remove NaC1 and allow subunit reassociation. The resulting LDHs were resolved by gel electrophoresis in the usual manner. RESULTS AND DISCUSSION
The muscle LDHs A detailed examination of Fig. 1 (rows M and m) illustrates the close similarity between muscle L D H s of lake and brook trout. For purposes of clarity the proposed subunit formulae of the lake trout series are given in small letters; the subunit formulae for brook trout muscle L D H s are written in capital letters. T h e A - B ( L D H 1-5) series in both species show quite similar, mobilities towards the anode. T h e lake trout 1-5 series shows activity usually skewed in favor of a4 or L D H - 1 . In the brook trout A-B series, the L D H activity is distributed binomially between the L D H s 1-5. All the components between L D H s 5-9 can be resolved in brook trout; in lake trout, it is unusual to find by our methods L D H s , 6 7 or 8 (b3cl, b2c~ and blcz), though L D H - 9 (ca) is clearly detectable. Always a certain amount of L D H activity migrates between L D H s 9 and 10; this is variable within and between species, for reasons which will be discussed below. L D H s 10-14 ( D - E series) in brook and lake trout have nearly identical electrophoretic mobilities. In view of these close similarities and the phylogenetic relationship between the two species, it is clear that each muscle L D H band in one is homologous to its counterpart in the other species. Spacings between the L D H s 10-14 indicate that these differ from each other by equal increments of charge. This is likewise true for the A - B and B - C series. However, the spacings between L D H s 10-14 are larger by a factor of 1.5 than those between, say, the L D H s 1-5. This result indicates independent components making up One complex isozyme system and such interpretation is further supported by the L D H patterns in liver tissue. Liver LDHs and the A, B and C subunits. Unlike muscle, liver in both species displays only 1 major L D H component, with 4 minor forms (usually undetectable
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by gel-staining methods) separating out at equal distances from each other. The number and charge properties of the liver L D H s fit the assumption of classical 2 subunit systems (Markert, 1963b), one subunit being produced very much in excess of the other; assembly is presumably random and yields 5 tetramers. As is evident in Fig. 1 (row 1 and L), however, the liver L D H s in lake trout are not the direct homologs of the liver L D H s in brook trout, a most striking observation in view of the close similarity of the muscle LDHs. Brook trout liver possesses L D H s 5-9, L D H - 9 (or C4) being in very high concentration, L D H s 5-8 appearing in lesser and lesser activity. In lake trout liver, the L D H 1-5 series is preponderant, L D H - 5 (or b4) having the highest activity, LDH-1 (or aA) having an activity so low it is also often undetectable by the gel-staining method (see Fig. 1, row L). Simultaneous 24 hr electrophoresis of liver and muscle L D H s from both brook and lake trout indicates that the main brook trout liver L D H and muscle CA (LDH-9) are indeed the same proteins, that the main lake trout liver L D H is identical to muscle LDH-5 or b 4 and so forth. That is to say, the liver and muscle systems are in fact homologous to each other and thus our use of similar nomenclature is probably justifiable. Tissue specificity derives from the relative abundance of different subunits, and, hence, different L D H proteins rather than from tissue-specific isozymes p e r se (see Markert, 1963a, for further discussion of this point). It is accepted that electrophoretic criterion of identity of different L D H s is not unequivocal. However, it is consistent with the data of Nissebaum et al. (1964) showing that mammalian L D H s of identical electrophoretie mobilities but of different tissues are apparently identical proteins. Presumably, then, each tissue in Salmonidae has the potentiality of synthesizing all of the subunits, though only in muscle is this potentiality expressed. Our interpretation of the trout liver L D H s predicts that L D H - 9 (C~, brook trout liver) and L D H - 5 (b4, lake trout liver) be homotetramers. Two kinds of evidence support the prediction: 1. Female lake trout x male brook trout crosses (called Splake) have been made and are available in abundance (Fry & Gibson, 1953). Now if the major liver L D H s are indeed "pure" tetramers (C Ain brook trout, b Ain lake trout), one outcome of the above cross would be the generation of 3 new kinds of LDHs, C3bl, C2b2 and Clb3, to yield 5 L D H s (Shaw, 1964). This result indeed is obtained (Fig. 2). Further details on interspecies inheritance will be published elsewhere. For the moment, it is gratuitous to our argument that when these 5 L D H s occur in Splake liver (middle row, Fig. 2), they occur in roughly a 1 : 4 : 6 : 4 : 1 ratio, that predicted by assuming a random assembly mechanism. This kind of distribution of L D H activity in the liver isozymes is not seen in either of the parental species (about 70 specimens of each species tested). 2. Since assembly of tetramers is thought to be epigenetic (Markert, 1963a), it should be and indeed is possible to generate the C3b ~, C~b 2 and Cab 3 isozymes in vitro. New LDHs, analogous to those seen in Splake liver, can be obtained by dissociation and reassociation of brook trout liver L D H and lake trout liver LDH.
BROOK
123456789 %
to C4
84
11
04
12
13
14 E4
FIG. 1. Resolution of trout LDHs on starch gel. Electrophoresis conditions: 18 hr at 35-40 mA and 200 V, in phosphate-citrate buffer, pH 7, ionic strength 0.52. Gel temperatures constant at lo-12°C. Activity per unit wet weight is approximately proportional to intensity of staining. Anode at left of all electrophoretograms. Origin marked by dark dashes. (1) Lake trout liver LDH. (m) Lake trout epaxial muscle LDH. (L) Brook trout liver LDH. (M) Brook trout muscle LDH. Subunit formulae for brook trout LDHs in capital letters; for lake trout LDHs, in small letters.
LIVER
CDH 64
oripn
FIG. 2. Liver LDHs of lake and brook trout and of one result of an F, hybrid between the two species (called Splake). Electrophoretic conditions as in Fig. 1. Time of run 18 hr. The liver LDH patterns for the hybrid trout (Splake) are a Out of about 70 brook and lake trout unique consequence of hybridization. examined, Splake kind of liver LDH patterns were never found.
A4
%Cl
*2=2
Al%
C4
origin I
ACC
1
A
A+
*4
64
=4
I
B
t
B+c
FI
origin
FIG. 3. Dissociation-reassembly experiments with A,, II, and C1 LDHs. Conditions of dissociation and reassociation given in text. Electrophoresis conditions as in Fig. 1. Time of electrophoresis, 15 hr. Subunit formulae given on the electrophoretogram.
FIG, 4. Comparison of LDH patterns in high speed supernatants of lake trout muscle homogenized in 0.88 M sucrose and distilled water. Electrophoresis condiI$ hr. LDHs 1-5 and LDH 9 (6, 7 tions as in Fig. 1. Time of el ec t rophoresis, and 8 not usually present in lake trout muscle) show a drop in activity in sucrose homogenates. Presumably, this drop in activity is due to removal during centrifugation of osmotically shrunk nuclei and/or mitochondria. LDHs lo-14 show Experiments with heart LDHs yielded similar essentially no change in activity. results.
MODEL FOR A COMPLEX L D H SYSTEM
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Fig. 3 (row B + C) shows that in one freeze-thaw cycle, dissociation and reassembly is incomplete but the C3bl, C2b~, Ctb 3 forms predicted by the model are clearly demonstrable. The generation of 3 new LDHs, in vivo by an interspecies cross, and in vitro by dissociation and reassociation of lake and brook trout liver LDHs, establishes that the main liver isozyme in each species is made up of one kind of subunit, and that 4 subunits aggregate to make up the enzyme protein. That is, C4 (LDH-9) and b 4 (LDH-5) are homotetramers (see Shaw, 1964, for discussion of limitations of this approach). On the assumption that tissue-specificity resides in isozyme expression and not in isozyme kind, we now can define unequivocally the 2 homologous isozymes in muscle L D H zymograms. The pertinent observation is that both C 4 (LDH-9) and B 4 (LDH-5) are within the muscle zymogram series (see Fig. 1, row M). A 3-subunit model based on random assembly yielding 15 LDHs allows only 1 "pure" tetramer, say B4, within the series; the other 2 "pure" tetramers must be terminal in zymograms, say anodally, A4, and cathodally, C 4. These experiments thus rule out the 3-subunit model (Goldberg, 1965; Markert & Faulhaber, 1965) as an explanation of multiple LDHs in lower vertebrates. Adams & Finnegan (1965) found that the presence of terminal bands tended to be variable, appearing at some stages in development but not at others. We have seen an analogous case in brain tissue LDHs of warm- and cold-adapted goldfish (Hochachka, 1965). It was therefore important to have evidence that the band which we label LDH-1 (A4) in this paper is indeed a homotetramer made of only A subunits. Three lines of evidence justify accepting LDH-1 as a "pure" tetramer: 1. If LDH-1 is made up of 1 subunit kind, subunit interactions with B would yield L D H patterns consistent with the bandings for brook and lake trout muscle and for lake trout liver (Fig. 1). 2. A-C interactions, when occurring, ought to likewise generate 3 new kinds of LDHs. In brook trout muscle and heart tissue, the gene for subunit B is frequently completely turned off, no B subunits being formed. In these cases, A and C subunits form the expected 5 LDHs, Aa, A3C 1.... , C a (Hochachka, unpublished). 3. If the assumption of a homotetramer is correct, dissociation-reassociation of LDH-1 should not, and does not, yield any new kinds of LDHs (Fig. 3, row A). To verify this we took advantage of S. gairdneri (rainbow trout) liver which possesses only a single L D H form, electrophoretically similar to brook trout LDH-1. Freeze-thaw treatment of homogenates with only LDH-1 activity does not generate new L D H forms on removal of salt (Fig. 3, row A). On the other hand, A-B and A-C interactions can be shown under identical circumstances to produce the predicted heteropolymers. Thus, mixtures of A4 and Ba yield on reassociation the expected 3 new forms, A3B1, A~B~ and A1B 3 (Fig. 3, row A+B). Similarly, mixtures of A4 and C4 produce 3 new forms, A3C1, AaC ~ and A1C 3 (Fig. 3, row A+C). These tests thus suggest a validity to the definition of LDH-1 as the homotetramer, A 4.
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P . W . Hocnncx-mn
Parenthetically, it is perhaps worth stressing that Fig. 3 represents results from one freeze-thaw cycle. It is evident that B-C hybridization is not complete but that A-B and A-C polymerizations have essentially gone to completion. This result is consistent with the L D H patterns in epaxial muscle, which show a greater abundance of A-B hybrids than of the B-C kind, but it does not account for the usual in vivo absence of A-C tetramers. The D - E system in trout muscle
If we accept the subunit model for LDHs, the fastest cathodal L D H within a series is presumably a "pure" tetramer; LDH-14 is thus assigned arbitrarily the subunit formula E 4. Anodal to LDH-14 (Fig. 1, row M) are 4 components separated by equal distances, but, as mentioned above, the interband spacings here are larger than in the L D H 1-9 group. (The disparity in spacings is clearer after 24 hr electrophoresis.) This suggests that the D - E series form an isozyme system largely independent of the A - B - C series and predicts that LDH-10 is a homopolymer to which we can assign the subunit formula D 4. Some amphibian L D H systems seem to include a set of components of this kind (Wright, 1965). Further work will be required to define these L D H s with greater confidence. In most zymograms, L D H activity between L D H - 9 and LDH-10 (between C 4 and D4) cannot be well resolved. This activity is not an artifact of the gel-staining procedure and is possibly formed by a certain amount of hybridization between the A - B - C group and the D and E subunits. Indeed, this sort of intermediate activity might be predicted by our model. Lack of resolution of the L D H between C 4 and D 4 and its variability between individuals presumably is related to the degree to which the mechanisms for non-random subunit assembly are not failsafe. That the assembly mechanism cannot be a random one is indicated by the development of 9 rather than 15 L D H s within the A - B - C complex. Were the A, B and C subunits to associate into tetramers by a random process, tissues like trout epaxial muscle would possess at least 20 LDHs, 15 in the A - B - C series plus the 5 in the D - E group. Reports approaching this number of isozymes have in fact been made (see Markert & Faulhaber, 1965; Wright, 1965) and may represent greater degrees of freedom in the assembly of L D H subunits than is evident in the trout tissues under consideration. Nature of constraints on subunit aggregation Akhough this area is largely unexplored, there are indications of at least two mechanisms potentially capable of restricting subunit assembly. One constraint appears to be related to cellular compartmentalization. Evidence from fractionation studies is difficult to obtain for LDH, which is typically very water-soluble (see, for example, Novikoff, 1960). In the usual kind of homogenates used in this study, intracellular organelles are osmotically swelled or disrupted. To overcome this problem, samples from a given muscle tissue were divided into equal wet weights and homogenates were prepared in either 0"88 M sucrose or distilled water. The
MODEL FOR A COMPLEX LDH SYSTEM
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usual procedure was then used in preparing the homogenates for gel electrophoresis. Centrifugation time and speeds were sufficiently high to spin down nuclei and mitochondria, but no attempt was made to fractionate the latter. L D H patterns of only high speed supernatants were assayed. After centrifugation of the sucrose homogenates (at about 30,000 g), there was a very noticeable drop in L D H s 1-5, a lesser disappearance of LDH-9, but essentially no change in the L D H s 10-14 when these were compared to L D H patterns from water supernatants (Fig. 4). The effect is not due to a specific sucrose inhibition for the L D H assay was performed after some 18 hr of electrophoresis in a gel medium which is free of sucrose. When similar tests were performed using lake trout heart, a highly aerobic tissue which possesses only an abundance of the A - B - C groups of L D H (Hochachka, unpublished), sucrose preparations yielded a very noticeable drop in L D H activity in all the isozymes to about the same extent. These preliminary experiments raise the probability of a cytoplasmic L D H system (the D - E group) separated from nuclear and/or mitochondrial ones (the A - B - C group), a situation possibly similar to that for malate dehydrogenases (Thorne et al., 1963). The presence in trout heart and brain of only A - B - C combinations (Hochachka, unpublished) might supply biological reasons for suspecting mitochondrial rather than nuclear associated activity. Since both nuclear and mitochondrial L D H activities are known to occur (see, for example, Novikoff, 1960), these preliminary tests do not allow a distinction to be made in the case of trout muscle. Tentatively, however, it appears that cellular compartmentalization may allow one means, a biological one, for restricting random subunit assortment. A second constraint relates to molecular size of the different LDHs. A few preliminary runs suggest that the fast anodal A-B tetramers (LDHs 1-5), assumed to be of the same molecular weight as other vertebrate LDHs, 140,000 g/mole (Fine et al., 1963; Daugherty, 1965), filter more rapidly than the D - E series through G-100 Sephadex and can be readily separated from the D - E L D H s in one run on a 50 cm column. The D - E tetramers filter at rates identical to hemoglobin and hence are tentatively assumed to be about one-half the molecular weight of the A-B LDHs. L D H - 9 (C4) seems to be intermediate in size. Appella (in Kaplan, 1964) has noted that the mammalian L D H "subunit" consists of two smaller components, each about 18,000 mol. wt. It is an interesting conjecture that the D - E series in trout muscle are tetramers formed from these smaller subunits. This entire question in the case of trout muscle L D H s requires much further work before confidence can be placed in these first experiments. They do, however, raise the possibility that size differences between the A-B and the D - E subunits affect monomer aggregation. This sort of mechanism, combined with other restrictions such as cellular compartmentalization, assures that assortment of the 5 subunits in trout muscle is quite unique rather than totally random. Dissociationreassociation tests show that also within the A - B - C group certain polymerizations seem to occur more readily than others. To what extent this might be due to size differences or to other more subtle structural restrictions is likewise a question which must be left open for future investigation.
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SUMMARY 1. By taking advantage of tissue and species homologies, a model involving 5 kinds of subunits is proposed to account for the multiple L D H s commonly found in lake and brook trout and perhaps other lower vertebrates. T h r e e subunits, A, B and C, generate a group of 9 anodally moving L D H s . L D H s 1-5 (the A - B group) can be written A4, AsB1, A2B2, A1B 3 and B 4. B and C polymerizations yield tetramer L D H s 6-9 (B3C 1, B2C2, B1C s and Ca). A - C interactions normally do not occur in vivo, but the expected A - C tetramers can be generated readily in dissociation-reassembly experiments. Similarly, the A-B and B-C heterotetramers can be formed in vitro. T h e dissociation-reassembly studies show that random assembly of the A, B and C subunits can yield 15 kinds of isozymes. T h e presence in brook and lake trout of only 9 forms implies that subunit assembly is at least partially directed. In cases where fewer constraints on subunit assembly occur, all 15 of the possible A - B - C tetramers would presumably be expressed. 2. D and E subunits are postulated to account for L D H s 10-14 (D4, D3E 1, D~E2, D1E 3 and E4), a group of 5 L D H s migrating near the origin at neutral pH. Since interband spacings, molecular weights and cellular location are probably different from L D H s 1-9, it is assumed that these form an essentially independent isozyme system. T h e r e is no evidence for D or E subunits interacting with the A, B or C monomers. 3. Probably all tissues are potentially capable of producing all 5 subunits, but only in muscle is this potentiality expressed in the species under study. Acknowledgements--Some facilities for this research were made possible through a grant from the National Research Council (Canada) to Dr. K. C. Fisher, University of Toronto. The development of the problem owes much to discussion with Geneva H. Williams. Part of the study was carried out while working as a postdoctoral fellow with Dr. W. L. Byrne, Department of Biochemistry, Duke University (U.S.P.H.S. Grant No. GM-06628-06S1).
REFERENCES ADAMS E. & FINNEGANC. V. (1965) An investigation of lactate dehydrogenase activity in early amphibian development. `7. exp. Zool. 158, 241-251. CHILSON O. P., COSTELLO L. A. • KAPLAN N. O. (1965) Studies on the mechanism of hybridization of lactic dehydrogenases in vitro. Biochemistry 4, 271-281. DAUGHERTY W. (1965) Analysis of the apparently single form of lactate dehydrogenase found in the flatfish, Paralichthys denatatus. Am. Zoologist 5, 205. (Abstract only.) FINE I. H., KAPLANN. O. & KUFrlNECD. (1963) Developmental changes of mammalian lactic dehydrogenases. Biochemistry 2, 116-121. FRY F. E. J. & GIBSONM. B. (1953) Lethal temperature experiments with speckled trout × lake trout hybrids..7. Heredity 44, 56-57. GOLDBERGE. (1965) Lactate dehygrodenases in trout: evidence for a third subunit. Science, N.Y. 148, 391-392. HOCHACHKAP. W. (1965) Isoenzymes in metabolic adaptation of a poikilotherm: Subunit relationships in lactic dehydrogenases of goldfish. Archs. Biochem. Biophys. 111, 96-103. KAPLAN N. O. (1964) Lactate dehydrogenase--Structure and function. In Subunit Structure of Proteins. Biochemical and Genetic Aspects, Brookhaven Symposia in Biology, No. 17, 131-153.
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KRAUSA. P. & NEELY C. L., JR. (1964) Human erythrocyte lactate dehydrogenase. Four genetically determined variants. Sdence, N . Y . 145, 595-597. MARKET C. L. (1963a) Epigenetic control of specific protein synthesis in differentiating cells. In Cytodifferentiation and Macromolecular Synthesis, 21st Symp. Soc. Study Dev. Growth (Edited by LOCKE, M.), pp. 64-84. Academic Press, New York. MARrmRT C. L, (1936b) Lactic dehydrogenase isozymes : dissociation and recombination of subunits. Science, N . Y . 140, 1329-1330. MAmC~T C. L. & FAULHAB~RI. (1965) Lactate dehydrogenase isozyme patterns of fish. jY. exp. Zool. 195, 319-332. NISSPBAVMJ. S., PACKERD. E. & BODANSHX"O. (1964) Comparison of the actions of human brain, liver and heart lactic dehydrogenase variants on nucleotide analogues and on substrate analogues in the absence and in the presence of oxalate and axamate..7, biol. Chem. 239, 2830-2834. NOVIHOFFA. B. (1960) Electron transport enzymes: biochemical and tetrazolium staining studies. In Histochemistry and Cytochemistry, Proc. 1st Int. Congr. 1960 (Edited by WEGMAN, R.), pp. 465-481. Macmillan, New York. SHAW C. R. (1964) The use of genetic variation in the analysis of isozyme structure. In Subunit Structure of Proteins. Biochemical and Genetic Aspects, Brookhaven Symposia in Biology, No. 17, 117-130. SMITHIES O. (1955) Zone electrophoresis in starch gels: group variations in the serum proteins of normal adults. Biochem. 3. 61, 629-641. THORNE C. J. R., GROSSMANL. I. & KAPLANN. O. (1963) Starch-gel electrophoresis of malate dehydrogenase. Biochem. biophys. Acta 73, 193-203. WRIGHT D. A. (1965) The development of lactate dehydrogenase isozymes in intraspecific crosses in the genus Rana. Am. Zoologist 5, 205. (Abstract only.) ZINKHAMW. H., BLANCOA. & KUPCYHKL. (1964) Lactate dehydrogenase in pigeon testes: genetic control by three loci. Science, N . Y . 144, 1353-1354.