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Lactate dehydrogenase isoenzymes and their genetic variation in coalfish (Gadus virens) and cod (Gadus morrhua)
Comp. Biochem. Physiol., 1970, Vol. 32, pp. 23 to 32. PergamonPress. Printed in Great Britain
LACTATE DEHYDROGENASE ISOENZYMES AND T H E I R GENETIC VARIATION IN COALFISH (GADUS VIRENS) AND COD (GADUS MORRHUA) I. E. L U S H Natural Environment Research Council, Fisheries Biochemical Research Unit, University of Aberdeen, Torry, Aberdeen AB1 3RA (Received 23 April 1969)
Abstract--1. The lactate dehydrogenase (L-lactate NAD oxidoreductase, E.C. 1.1.1.27) isoenzymes in extracts of several tissues of coalfish (Gadus virens) and cod (Gadus morrhua) were examined by starch-gel electrophoresis in two buffer systems. 2. In both species three principal lactate dehydrogenase polypeptides occur. The H and L polypeptides, which are present in several tissues, combine to form homotetramers and heterotetramers. The K polypeptide, which occurs mainly in white skeletal muscle, does not combine with the other two polypeptides. 3. Coalfish is polymorphic with respect to the K polypeptide and cod is polymorphic with respect to the H polypeptide. No differences in gene frequencies have been found between different populations within either species. 4. The effect of different buffer systems on the separation of the lactate dehydrogenase isoenzymes of different species was investigated.
INTRODUCTION MOST of the early work done on the isoenzymes of vertebrate lactate dehydrogenase ( L D H ) was restricted to mammals and birds (Kaplan & Goodfriend, 1954); however, the results which are now beginning to accumulate show clearly that fish species vary m u c h more in the degree of complexity of their L D H isoenzymes than do warm-blooded vertebrates (Markert & Faulhaber, 1965). Many fiat fish (Heterosomata) have only one L D H isoenzyme in all somatic tissues except brain and retina (Daugherty, 1965 ; Lush et al., 1969), whereas the tissues of the brook trout and lake trout (Salvelinusfontinalis and S. namaycush) contain a complicated series of isoenzymes composed of at least five different kinds of polypeptide (Morrison & Wright, 1966). T h e comparison of the arrays of L D H isoenzymes, and their component polypeptides and underlying genetic loci, in different groups and species of vertebrates seems likely to develop into an interesting exercise in comparative biochemical genetics. * Present address: Biology Department, Royal Free Hospital School of Medicine, University of London, 8 Hunter Street, London W.C.1. 23
24
I.E. LUSH
As part of a programme of work in this laboratory on the biochemical and genetical aspects of L D H isoenzymes in fish, I have made a search for a species in which the skeletal muscle contains predominantly a single L D H isoenzyme which is also genetically polymorphic. It was hoped that such a species would provide suitable material for the detailed biochemical comparison of polymorphic variants of L D H . Muscle extracts from fish of eight local species have been examined by starch-gel electrophoresis. Only in coalfish (Gadus virens L.) was polymorphism found. Fortunately, the muscle L D H of this species consists mainly of one isoenzyme. Odense et al. (1966) have shown that an L D H polymorphism exists in cod (Gadus morrhua L.) off the Atlantic coast of Canada, but the polymorphic polypeptide is not strongly represented in skeletal muscle. T h e present paper describes the coalfish polymorphism and also shows that the polymorphism which was found by Odense et al. (1966) is present in cod caught within 40 miles of Aberdeen, Scotland. T h e mol. wt. of L D H in many vertebrate species is about 140,000 (Pesce et al., 1967), and it now seems probable that in all vertebrates the L D H molecule is a tetramer of four polypeptides each with a mol. wt. of 35,000. I shall refer to an L D H tetramer which is composed of four indistinguishable polypeptides as a homotetramer. T h e term "heterotetramer" will be used when the component polypeptides are not all identical, either because of genetic polymorphism or because of the co-existence of more than one L D H locus in the same genome. It has been found by several different authors (Fritz & Jacobson, 1965; Stambaugh & Buckley, 1967) that starch-gel zymograms of L D H often show a n u m b e r of subsidiary isoenzymes in addition to those which can be explained in terms of different combinations of the polypeptides thought to be present in the tissue under examination. It is supposed that these supernumerary isoenzymes may be formed by mutual rearrangements of the polypeptide chains within each tetramer, or alternatively by the binding of smaller organic or inorganic molecules. Whatever their causation may be, I shall refer to isoenzymes attributable to such secondary factors as "satellite" isoenzymes. MATERIALS AND METHODS The coalfish were routinely obtained from Allan and Dey Ltd., Wholesale Fish Merchants, Aberdeen. They were 3-7 days post-mortem but had been gutted at sea and kept on ice. The sex and age of the coalfish were not known but all were adults of at least 45 cm in length. The general area in which they had been caught was known, but not the exact position. The cod, all adults, were either locally caught fish obtained fresh from Mr. T. Wilson, Wholesale Fish Merchant, Aberdeen, or were freshly killed individuals which had been kept in the Unit's aquarium for several days. With both species the LDH zymograms obtained from the commercially obtained material were identical to those of the freshly killed fish. Tissues were homogenized in cold 1% saline in a "TRI-R" Potter-Elvejhem homogenizer, centrifuged at 20,000 g for 30 min and the supernatant used for electrophoresis. In the survey of coalfish muscle phenotypes, some fish were typed by absorbing the juice expressed from the cut muscle directly into rectangles of Whatman No. 1 filter paper which were then inserted into the gel as described below.
C O A L F I S H AND COD
LDH
25
VARIATION
Starch gels for electrophoresis were made with starch from Connaught Laboratories, Toronto. The buffer systems of Poulik (1957), Shaw & Barto (1963) and Syner & Goodman (1966) were used. The gels were cooled to 4°C before use and the samples absorbed into rectangles of Whatman 3-ram paper and inserted into the cooled gel. Electrophoresis was carried out horizontally for about 17 hr at 4°C with stabilized voltage gradients along the gel of 4'0 V/cm (Poulik, 1957) and 6"0V/era (Syner & Goodman, 1966). After electrophoresis, each sliced gel was incubated, cut surface uppermost, in darkness at 37°C in 0.05 M TrisHC1 buffer, pH 8-0, containing reagents at the following concentrations: Lithium L-lactate, 45"0 mM; NAD, 30"0 mg/100 ml; N-methylphenozonium methosulphate, 2-0 mg/100 ml; Nitro-blue tetrazolium, 5"0 rag/100 ml. When the substrate was omitted none of the LDH bands appeared. Lithium DL-lactate, NAD and nitro-blue tetrazolium was obtained from Sigma Chemical Co. Ltd., London. All other reagents were of analytical grade. RESULTS
Coalfish Coalfish from the west coast of Scotland and from fishing grounds round the Faeroes and Iceland were surveyed with respect to the L D H zymograms obtained from extracts of white skeletal muscle. In Syner & Goodman gels, each fish could be classified as one of the three phenotypes illustrated in Fig. 1. Clearly, there is a genetic polymorphism of the major skeletal muscle L D H isoenzyme in this species. On the reasonable assumption that coalfish L D H is a tetramer, it seems clear that the individuals of phenotypes LdhK-F and LdhK-S, which both have single major isoenzymes near the centre of the zymogram, are each homozygous for a different allele at the locus which determines the electrophoretic mobility, and probably also the structure, of the polypeptide of which this tetramer is composed. This locus will be referred to as LdhK. According to this scheme, the single isoenzyme of the LdhK-F phenotype is a homotetramer which can be written as K4r and similarly the single isoenzyme of the LdhK-S phenotype is K s. This is borne out by the pattern of the zymogram of the presumptive heterozygous phenotype, LdhK-FS in which the five equally spaced isoenzymes occur in the positions and relative amounts (as judged by eye from the zymogram) to be expected from a random combination in tetramers of an equal number of the two polypeptides K r and K s. Thus the composition of the five isoenzymes of the heterozygoteis assumed to be K~, K 3FKt, S K r2 K,, S K 1r K s3 and K s. The symbolism used can be summarized as follows:
Genotype Ldh~:/Ldh~: Ldh~c/Ldhs LdhS/Ldh s
Phenotype LdhK-F LdhK-FS Ldh~:-S
Polypeptides synthesized Ks K s and K s Ks
Table 1 shows that the polymorphism exists in all three sea areas sampled. The phenotype numbers accord well with Hardy-Weinberg expectations, and the allele frequencies are identical in the samples from each area. A minor isoenzyme can be seen near the anodic end of the zymogram of the muscle extracts in Fig. 1. Since this isoenzyme is unaffected by the K polypeptide variation, presumably the K polypeptide does not enter into its composition and
26
I.E. LUSH
TABLE 1--NUMBERS OF COALFISHAND COD OF EACH LDH PHENOTYPE, COMPAREDWITH NUMBERS EXPECTEDF R O M T H E HARDY--WEINBERGDISTRIBUTION Species
Location
Allele Totals frequencies
LDH phenotypes Ldh K
Ldh s
I
Coalfish
Scottish west coast Faeroe Iceland
S Observed: 104 Calculated: 102-06 Observed: 85 Calculated: 84"24 Observed: 20 Calculated : 20
I
Cod
Aberdeen
Observed: Calculated :
S 35 27"78
SF 19 22"68 17 18"72 5 5 Ldh~
F 3 1"26 2 1"04 0 0
SF 73 87"58
F 76 68"64
I I
126
0"90
104
0'90
25
0"90 Ldh s
I
184
0-39
we can for the moment assume that it is a homotetramer of another polypeptide, H, which is determined by a second locus, L d h n . No intermediate isoenzymes are found between H 4 and K4, indicating that heterotetramers of mixed H and K composition do not occur in skeletal muscle. In extracts of heart muscle H 4 is the major isoenzyme although the polymorphic K 4 isoenzyme is clearly present. In heart extracts two minor bands which appear consistently between K 4 and H a are indicated in Fig. 1B by square brackets. T h e y may be due to the combination of three K polypeptides with one other polypeptide, but in the LdhK-F phenotype the minor band lies closely adjacent to the K~ isoenzyme, which would not be its expected position if it were KaH1. Retina extracts are still more complex in that, in addition to the K 4 isoenzymes, which are just visible, a series of four new bands spans the distance between H 4 and the origin. These can be interpreted as the result of the presence of a third polypeptide, L, determined by a locus L d h L. T h e isoenzyme near the origin is accordingly the homotetramer L 4 and the three intermediate isoenzymes are heterotetramers formed by combinations of H and L as indicated in Fig. tB. T h e r e is no detectable combination between the L and K polypeptides in retina and the polymorphism has no effect on the isoenzymes formed by combinations of H and L. In all three tissues from Ldhr~-F coalfish a minor band corresponding in mobility to K s is present and is indicated in Fig. 1B by curved brackets. This may be a homotetramer of yet another polypeptide which is present in all tissues and happens to coincide with K s. T h e retinas of twenty-one coalfish gave no evidence of genetic variation of the H or L polypeptides. T h e hearts of another four fish showed no variation of the H polypeptide.
-K -K -K -K K
,~ Heart
Retina
A_
Muscle
(A) Photograph and (B) diagram of LDH zymograms of heart, retina and white skeletal muscle from coalfish of the three Ldh, phenotypes (Syner & Goodman gel). Samples 1, 4 and 7 are from a coalfish of type Ldh,-F. Samples 2, 5 and 8 are from type LdhK-FS. Samples 3, 6 and 9 are from type Ldh,-S. FIG.
1.
F
FS
S
F
S
FS
F
FS
S
0
”
..)
( i
/;
-’
:_
,.
,_’
-,
” -T-
L4
B
HWXi FIG. 4. (A) Photograph white skeletal muscle
Retina
Muscle
and (R) diagram of LDH zymograms of heart, retina and from cod of each of the Ldh, phenotypes (Poulik gel).
COALFISH
AND
COD
LDH
VARIATION
27
Other coalfish tissues contain a selection of the LDH isoenzymes described above (Fig. 2). Liver contains a predominance of L, and much less of the other two homotetramers. The absence of the HL heterotetramers in liver contrasts
FIG. 2. Diagram of zymograms of different tissues from a type Ldh,-S coalfish (Syner & Goodman gel). The tissues were: 1, retina; 2, red skeletal muscle; 3, liver; 4, spleen; 5, gill; 6, brain ; 7, erythrocytes; 8, gas gland.
with their presence in retina, spleen and erythrocytes. The red skeletal muscle, which lies under the lateral line system, differs from white skeletal muscle and resembles the heart in that the H, isoenzyme is stronger than K,. Red skeletal muscle and gill tissue also contain the minor isoenzyme which is present in heart muscle. The highly vascular gas gland of the swim bladder was expected to show some unique or unusual LDH zymogram because the production of gas by this tissue for hydrostatic purposes is thought to involve the secretion of lactic acid (Alexander, 1966); however, it showed only K, and H,. The polymorphism of the K polypeptide was apparent in every tissue. Testis was not investigated. Before it was found that Syner & Goodman gels give such a clear separation of K, from H,, most of the coalfish referred to in Table 1 had already been typed in Poulik gels. Figure 3 shows that in this gel the K, isoenzymes have mobilities similar to the H, isoenzyme; moreover, the K, homotetramers are accompanied by two satellite bands. Although the relative positions of the K, and H, isoenzymes are so different in the two kinds of gel the L, isoenzymes of liver, erythrocyte and retina correspond exactly in both gels, which confirms their identity. The identity of H, in different tissues was similarly confirmed. It is clearly helpful to examine complex mixtures of LDH isoenzymes in more than one buffer system, a point which will be reinforced in the following section on cod. A consistent but unexplained finding in the Poulik gels was the tendency of the H,L, isoenzyme in all three phenotypes to split into two bands, the slower one being the weaker. This effect was noticed with the corresponding cod
28
I. E.
LUSH
isoenzyme in both types of gel (see below). Syner & Goodman (1966) found similar satellite bands on the anodal side of the H,M, isoenzyme of the gelada baboon LDH.
F
FS
s
F
FS
s
F
FS
S
h
.,
-_
FIG. 3.
Diagram of LDH zymograms of heart, retina and white skeletal muscle from coalfish of the three Ldh, phenotypes (Poulik gel). Samples as in Fig. 1.
Cod Odense et al. (1966, 1969) h ave described an LDH polymorphism in samples of cod which were caught off the Atlantic coast of Canada. They found three common phenotypes which could be recognized by starch-gel electrophoresis of a variety of tissues including heart and retina. I have examined tissues from 184 cod caught within a distance of 40 miles of Aberdeen. Most of these were typed from the zymograms of retinal extracts in Poulik gels. The polymorphism described by Odense et al. (1966) was clearly recognizable, in fact the zymograms appeared to be very similar to theirs even although they used a different buffer system for electrophoresis. The correspondence was checked by comparing, in the same Poulik gel, extracts of retinas of each phenotype supplied by Dr. Odense with extracts of retinas of locally caught cod. The three phenotypes appeared to be the same in each population, confirming that we were dealing with the same polymorphism in Scottish cod as in Canadian cod. The LDH zymograms obtained in a Poulik gel with extracts of heart, retina and white skeletal muscle from cod of each phenotype are shown in Fig. 4. In muscle, the fast major isoenzyme predominates and showed no sign of genetic variation in forty individuals. It can be assumed to be a homotetramer of a K polypeptide determined by a locus homologous with the polymorphic Ldh, locus
COALFISH AND COD
LDH
VARIATION
29
of coalfish. The K, isoenzyme of cod, like that of coalfish in Poulik gels, is accompanied by satellite bands in front and behind. Farther back are traces of the genetically variable isoenzymes which form the predominant isoenzymes of heart. The three cod phenotypes are clearly due to variation of the H polypeptide and they will therefore be designated Ldhn-F, Ldhn-FS and Ldh,-S. The zymogram of the heart of the Ldhn-FS cod shows the five equally spaced bands which are characteristically found when two different polypeptides combine The heart zymograms of Ldhn-F and together at random to form tetramers. Ldhn-S cod have major isoenzymes which correspond, respectively, with the fastest and slowest isoenzymes of the heterozygote. The two corresponding alleles at the Ldh, locus will be referred to as Ldhg and Ldh$. The numbers of each phenotype found in a total of 184 cod caught between December 1966 and June 1968 are shown in Table 1. There is a slight but significant excess in the proportion of homozygotes compared with the Hardy-Weinberg expectations (x2 = 5.08, P
30
I. E. LUSH
in a Syner & Goodman gel. The zymogram of muscle shows K, and a small amount of the polymorphic H,, although the H, isoenzymes are hardly visible in the heterozygote. In the retinas the K, isoenzyme falls in the same place as in muscle, although it happens to coincide with other retinal isoenzymes. The retinal array of isoenzymes formed by combinations of L and the polymorphic H polypeptides is less clearly defined than in the Poulik gel but can nevertheless be seen to give the same phenotypes, F
FS
s
F
FS
S
F
FS
FIG. 5. Diagram of zymograms of heart, retina and white cod of each of the Ldh, phenotypes (Syner & Goodman gel).
S
skeletal muscle from Samples as in Fig. 4.
In Fig. 5 the heart zymograms of the three phenotypes show the expected H, patterns and the isoenzyme K, is also present in each. However, the bands enclosed in square brackets in Fig. 5 have no counterparts in the Pouiik zymogram and as yet they have no firm explanation in terms of polypeptide composition. The pattern of these anomalous isoenzymes in the three phenotypes suggests that they are heterotetramers made up of two H polypeptides combined with two polypeptides of a much slower mobility. We may symbolize these isoenzymes by H,Q,. In fact in a number of experiments in which the length of the run or the composition of the Syner & Goodman buffer were altered slightly it was found that the H,Q, isoenzymes always correspond exactly with the retinal H,L, isoenzymes of the corresponding phenotype. However, if the anomalous isoenzymes really are H,L, why do they not appear in heart zymograms in Poulik
COALFISH AND COD
LDH
VARIATION
31
gels ? It would also be of interest to know if they are in some way comparable to the anomalous (square bracketed) isoenzymes of coalfish shown in Fig. 1, although from the genetical evidence this seems unlikely. The distribution of LDH isoenzymes in other cod tissues broadly resembles that found in coalfish. When the LDH isoenzymes of a coalfish and an Ldhn-F cod were compared in the same gels, it was found that the mobilities of the five isoenzymes of the HL series were the same in the two species. This was true for each of the two buffer systems. However Dr. P. Odense (personal communication) has evidence which may show that the L4 isoenzymes of cod liver and retina are electrophoretically separable under some conditions. DISCUSSION
One conclusion from this survey of cod and coalfish LDH is that both species have three principal LDH loci active in their somatic tissues. The comparable loci in the two species are so similar in their tissue-specific expression that they can be assumed to be homologous. In terms of the H, L and K nomenclature adopted above, one finds that the H and L polypeptides combine together to form heterotetramers but there is no clear evidence of combination between K and the other two. In cod the H locus is polymorphic while in coalfish the K locus is polymorphic. Another conclusion is that, because of the unpredictable occurrence of tissuespecificity and genetic variation, it is unwise to arrive at an estimate of the number of LDH loci in the genome of a fish species without a survey of different tissues and a number of individuals. The comparison of zymograms obtained with different buffer systems will add conviction to the estimate. In both cod and coalfish the LDH zymogram of red skeletal muscle resembles that of the heart and differs from that of the white skeletal muscle in the predominance of H, over K,. Fish red skeletal muscle is used for slow and steady cruising over long periods, while the white muscle comes into use for rapid movements of short duration (Bone, 1966). Th e energy metabolism of red muscle is mainly aerobic while white muscle is rapidly fatigued, presumably by the accumulation of an oxygen debt. This is consistent with the finding that red muscle resembles heart muscle in its LDH zymogram. With regard to the Ldh gene frequencies, the data have not produced any surprises. The frequencies in the three coalfish populations are obviously very similar, and the gene frequency of the local Aberdeen cod population is almost exactly the same as in cod off the Atlantic coast of Canada which Odense et al. (1966) found to have an Ldhg frequency between 0.35 and O-39. The large differences between the haemoglobin gene frequencies in Scottish and Canadian cod (O-65 and O-04, respectively for the H6P allele), as well as other biological considerations, shows that the two populations are entirely separate (Sick, 1965). It would be of interest to see if the cod LDH polymorphism, unlike the hemoglobin polymorphism, is maintained at the same gene frequency throughout the range of the species. 2
I. E.
32
LUSH
Acknowledgements-My thanks are due to Dr. P. T. Grant for his help and encouragement, and to Mr. D. Knox for technical assistance.
REFERENCES ALEXANDERR. McN. (1966) Physical aspects of swimbladder function. BioE. Rev. 41, 141-176. BONE Q. (1966) On the function of the two types of myotomal muscle fibre in elasmobranch fish. J. mar. biol. Ass. U.K. 46, 321-349. DAUGHERTYW. (1965) Analysis of the apparently single form of lactate dehydrogenase (LDH) found in the flatfish, Paralichthys dentatus. Am. Zool. 5, 20.5. FRITZ P. J. & JACOBSONK. B. (1965) Multiple molecular forms of lactate dehydrogenase. Biochemistry 4, 282-289. KAPLAN N. 0. & GOODFRIENDT. L. (1964) Role of the two types of lactic dehydrogenase. Adv. Enz. Regul. 2, 203-212. LUSH I. E., COWEY C. B. & KNOX D. (1969) The lactate dehydrogenase isoenzymes of twelve species of flatfish (Heterosomata). J. exp. Zool. (In press.) MARKERT C. L. & FAULHABERI. (1965) Lactate dehydrogenase isozyme patterns of fish. J. exp. Zool. 159, 319-332. MORRISON W. J. & WRIGHT J. E. (1966) G enetic analysis of three lactate dehydrogenase isozyme systems in trout: evidence for linkage of genes coding subunits A and B. J. exp. 2001. 163, 259-270. ODENSE P. H., LEUNG T. C. & ALLEN T. M. (1966) An electrophoretic study of tissue proteins and enzymes of four Canadian cod populations. Cons. Perm. Int. Explor. Mer. C.M. No. G, 14, 1. ODENSEP. H., LEUNG T. C. & ALLEN T. M. (1969) Multiple forms of lactic dehydrogenase in the cod. Biochem. Genetics 3, 317-324. PESCE A., FONDY T. P., STOLZENBACHF., CASTILLO F. & KAPLAN N. 0. (1967) The comparative enzymology of lactic dehydrogenase-III. Properties of the H, and M, enzymes from a number of vertebrates. J. biol. Chem. 242, 2151-2167. POULIK M. D. (1957) Starch gel electrophoresis in a discontinuous system of buffers. Nature, Lond. 180, 1477. SHAW C. R. & BARTOE. (1963) Genetic evidence for the subunit structure of lactate dehydrogenase isozymes. Proc. natn Acad. Sci. U.S.A. 50, 211-214. SICK K. (1965) Haemoglobin polymorphism of cod in the North Sea and the South Atlantic Ocean. Hereditas 54, 49-73. STAMBAUGHR. & BUCKLEY J. (1967) The enzymic and molecular nature of the lactic dehydrogenase subbands and X4 isoenzyme. r. biol. Chem. 242, 40534059. SYNER F. N. & GOODMANM. (1966) Polymorphism of lactate dehydrogenase in gelada baboons. Science 151, 206-208. Key
Word Index-Coalfish
; cod ; isoenzyme ; lactate dehydrogenase;
polymorphism.
LACTATE DEHYDROGENASE ISOENZYMES AND T H E I R GENETIC VARIATION IN COALFISH (GADUS VIRENS) AND COD (GADUS MORRHUA) I. E. L U S H Natural Environment Research Council, Fisheries Biochemical Research Unit, University of Aberdeen, Torry, Aberdeen AB1 3RA (Received 23 April 1969)
Abstract--1. The lactate dehydrogenase (L-lactate NAD oxidoreductase, E.C. 1.1.1.27) isoenzymes in extracts of several tissues of coalfish (Gadus virens) and cod (Gadus morrhua) were examined by starch-gel electrophoresis in two buffer systems. 2. In both species three principal lactate dehydrogenase polypeptides occur. The H and L polypeptides, which are present in several tissues, combine to form homotetramers and heterotetramers. The K polypeptide, which occurs mainly in white skeletal muscle, does not combine with the other two polypeptides. 3. Coalfish is polymorphic with respect to the K polypeptide and cod is polymorphic with respect to the H polypeptide. No differences in gene frequencies have been found between different populations within either species. 4. The effect of different buffer systems on the separation of the lactate dehydrogenase isoenzymes of different species was investigated.
INTRODUCTION MOST of the early work done on the isoenzymes of vertebrate lactate dehydrogenase ( L D H ) was restricted to mammals and birds (Kaplan & Goodfriend, 1954); however, the results which are now beginning to accumulate show clearly that fish species vary m u c h more in the degree of complexity of their L D H isoenzymes than do warm-blooded vertebrates (Markert & Faulhaber, 1965). Many fiat fish (Heterosomata) have only one L D H isoenzyme in all somatic tissues except brain and retina (Daugherty, 1965 ; Lush et al., 1969), whereas the tissues of the brook trout and lake trout (Salvelinusfontinalis and S. namaycush) contain a complicated series of isoenzymes composed of at least five different kinds of polypeptide (Morrison & Wright, 1966). T h e comparison of the arrays of L D H isoenzymes, and their component polypeptides and underlying genetic loci, in different groups and species of vertebrates seems likely to develop into an interesting exercise in comparative biochemical genetics. * Present address: Biology Department, Royal Free Hospital School of Medicine, University of London, 8 Hunter Street, London W.C.1. 23
24
I.E. LUSH
As part of a programme of work in this laboratory on the biochemical and genetical aspects of L D H isoenzymes in fish, I have made a search for a species in which the skeletal muscle contains predominantly a single L D H isoenzyme which is also genetically polymorphic. It was hoped that such a species would provide suitable material for the detailed biochemical comparison of polymorphic variants of L D H . Muscle extracts from fish of eight local species have been examined by starch-gel electrophoresis. Only in coalfish (Gadus virens L.) was polymorphism found. Fortunately, the muscle L D H of this species consists mainly of one isoenzyme. Odense et al. (1966) have shown that an L D H polymorphism exists in cod (Gadus morrhua L.) off the Atlantic coast of Canada, but the polymorphic polypeptide is not strongly represented in skeletal muscle. T h e present paper describes the coalfish polymorphism and also shows that the polymorphism which was found by Odense et al. (1966) is present in cod caught within 40 miles of Aberdeen, Scotland. T h e mol. wt. of L D H in many vertebrate species is about 140,000 (Pesce et al., 1967), and it now seems probable that in all vertebrates the L D H molecule is a tetramer of four polypeptides each with a mol. wt. of 35,000. I shall refer to an L D H tetramer which is composed of four indistinguishable polypeptides as a homotetramer. T h e term "heterotetramer" will be used when the component polypeptides are not all identical, either because of genetic polymorphism or because of the co-existence of more than one L D H locus in the same genome. It has been found by several different authors (Fritz & Jacobson, 1965; Stambaugh & Buckley, 1967) that starch-gel zymograms of L D H often show a n u m b e r of subsidiary isoenzymes in addition to those which can be explained in terms of different combinations of the polypeptides thought to be present in the tissue under examination. It is supposed that these supernumerary isoenzymes may be formed by mutual rearrangements of the polypeptide chains within each tetramer, or alternatively by the binding of smaller organic or inorganic molecules. Whatever their causation may be, I shall refer to isoenzymes attributable to such secondary factors as "satellite" isoenzymes. MATERIALS AND METHODS The coalfish were routinely obtained from Allan and Dey Ltd., Wholesale Fish Merchants, Aberdeen. They were 3-7 days post-mortem but had been gutted at sea and kept on ice. The sex and age of the coalfish were not known but all were adults of at least 45 cm in length. The general area in which they had been caught was known, but not the exact position. The cod, all adults, were either locally caught fish obtained fresh from Mr. T. Wilson, Wholesale Fish Merchant, Aberdeen, or were freshly killed individuals which had been kept in the Unit's aquarium for several days. With both species the LDH zymograms obtained from the commercially obtained material were identical to those of the freshly killed fish. Tissues were homogenized in cold 1% saline in a "TRI-R" Potter-Elvejhem homogenizer, centrifuged at 20,000 g for 30 min and the supernatant used for electrophoresis. In the survey of coalfish muscle phenotypes, some fish were typed by absorbing the juice expressed from the cut muscle directly into rectangles of Whatman No. 1 filter paper which were then inserted into the gel as described below.
C O A L F I S H AND COD
LDH
25
VARIATION
Starch gels for electrophoresis were made with starch from Connaught Laboratories, Toronto. The buffer systems of Poulik (1957), Shaw & Barto (1963) and Syner & Goodman (1966) were used. The gels were cooled to 4°C before use and the samples absorbed into rectangles of Whatman 3-ram paper and inserted into the cooled gel. Electrophoresis was carried out horizontally for about 17 hr at 4°C with stabilized voltage gradients along the gel of 4'0 V/cm (Poulik, 1957) and 6"0V/era (Syner & Goodman, 1966). After electrophoresis, each sliced gel was incubated, cut surface uppermost, in darkness at 37°C in 0.05 M TrisHC1 buffer, pH 8-0, containing reagents at the following concentrations: Lithium L-lactate, 45"0 mM; NAD, 30"0 mg/100 ml; N-methylphenozonium methosulphate, 2-0 mg/100 ml; Nitro-blue tetrazolium, 5"0 rag/100 ml. When the substrate was omitted none of the LDH bands appeared. Lithium DL-lactate, NAD and nitro-blue tetrazolium was obtained from Sigma Chemical Co. Ltd., London. All other reagents were of analytical grade. RESULTS
Coalfish Coalfish from the west coast of Scotland and from fishing grounds round the Faeroes and Iceland were surveyed with respect to the L D H zymograms obtained from extracts of white skeletal muscle. In Syner & Goodman gels, each fish could be classified as one of the three phenotypes illustrated in Fig. 1. Clearly, there is a genetic polymorphism of the major skeletal muscle L D H isoenzyme in this species. On the reasonable assumption that coalfish L D H is a tetramer, it seems clear that the individuals of phenotypes LdhK-F and LdhK-S, which both have single major isoenzymes near the centre of the zymogram, are each homozygous for a different allele at the locus which determines the electrophoretic mobility, and probably also the structure, of the polypeptide of which this tetramer is composed. This locus will be referred to as LdhK. According to this scheme, the single isoenzyme of the LdhK-F phenotype is a homotetramer which can be written as K4r and similarly the single isoenzyme of the LdhK-S phenotype is K s. This is borne out by the pattern of the zymogram of the presumptive heterozygous phenotype, LdhK-FS in which the five equally spaced isoenzymes occur in the positions and relative amounts (as judged by eye from the zymogram) to be expected from a random combination in tetramers of an equal number of the two polypeptides K r and K s. Thus the composition of the five isoenzymes of the heterozygoteis assumed to be K~, K 3FKt, S K r2 K,, S K 1r K s3 and K s. The symbolism used can be summarized as follows:
Genotype Ldh~:/Ldh~: Ldh~c/Ldhs LdhS/Ldh s
Phenotype LdhK-F LdhK-FS Ldh~:-S
Polypeptides synthesized Ks K s and K s Ks
Table 1 shows that the polymorphism exists in all three sea areas sampled. The phenotype numbers accord well with Hardy-Weinberg expectations, and the allele frequencies are identical in the samples from each area. A minor isoenzyme can be seen near the anodic end of the zymogram of the muscle extracts in Fig. 1. Since this isoenzyme is unaffected by the K polypeptide variation, presumably the K polypeptide does not enter into its composition and
26
I.E. LUSH
TABLE 1--NUMBERS OF COALFISHAND COD OF EACH LDH PHENOTYPE, COMPAREDWITH NUMBERS EXPECTEDF R O M T H E HARDY--WEINBERGDISTRIBUTION Species
Location
Allele Totals frequencies
LDH phenotypes Ldh K
Ldh s
I
Coalfish
Scottish west coast Faeroe Iceland
S Observed: 104 Calculated: 102-06 Observed: 85 Calculated: 84"24 Observed: 20 Calculated : 20
I
Cod
Aberdeen
Observed: Calculated :
S 35 27"78
SF 19 22"68 17 18"72 5 5 Ldh~
F 3 1"26 2 1"04 0 0
SF 73 87"58
F 76 68"64
I I
126
0"90
104
0'90
25
0"90 Ldh s
I
184
0-39
we can for the moment assume that it is a homotetramer of another polypeptide, H, which is determined by a second locus, L d h n . No intermediate isoenzymes are found between H 4 and K4, indicating that heterotetramers of mixed H and K composition do not occur in skeletal muscle. In extracts of heart muscle H 4 is the major isoenzyme although the polymorphic K 4 isoenzyme is clearly present. In heart extracts two minor bands which appear consistently between K 4 and H a are indicated in Fig. 1B by square brackets. T h e y may be due to the combination of three K polypeptides with one other polypeptide, but in the LdhK-F phenotype the minor band lies closely adjacent to the K~ isoenzyme, which would not be its expected position if it were KaH1. Retina extracts are still more complex in that, in addition to the K 4 isoenzymes, which are just visible, a series of four new bands spans the distance between H 4 and the origin. These can be interpreted as the result of the presence of a third polypeptide, L, determined by a locus L d h L. T h e isoenzyme near the origin is accordingly the homotetramer L 4 and the three intermediate isoenzymes are heterotetramers formed by combinations of H and L as indicated in Fig. tB. T h e r e is no detectable combination between the L and K polypeptides in retina and the polymorphism has no effect on the isoenzymes formed by combinations of H and L. In all three tissues from Ldhr~-F coalfish a minor band corresponding in mobility to K s is present and is indicated in Fig. 1B by curved brackets. This may be a homotetramer of yet another polypeptide which is present in all tissues and happens to coincide with K s. T h e retinas of twenty-one coalfish gave no evidence of genetic variation of the H or L polypeptides. T h e hearts of another four fish showed no variation of the H polypeptide.
-K -K -K -K K
,~ Heart
Retina
A_
Muscle
(A) Photograph and (B) diagram of LDH zymograms of heart, retina and white skeletal muscle from coalfish of the three Ldh, phenotypes (Syner & Goodman gel). Samples 1, 4 and 7 are from a coalfish of type Ldh,-F. Samples 2, 5 and 8 are from type LdhK-FS. Samples 3, 6 and 9 are from type Ldh,-S. FIG.
1.
F
FS
S
F
S
FS
F
FS
S
0
”
..)
( i
/;
-’
:_
,.
,_’
-,
” -T-
L4
B
HWXi FIG. 4. (A) Photograph white skeletal muscle
Retina
Muscle
and (R) diagram of LDH zymograms of heart, retina and from cod of each of the Ldh, phenotypes (Poulik gel).
COALFISH
AND
COD
LDH
VARIATION
27
Other coalfish tissues contain a selection of the LDH isoenzymes described above (Fig. 2). Liver contains a predominance of L, and much less of the other two homotetramers. The absence of the HL heterotetramers in liver contrasts
FIG. 2. Diagram of zymograms of different tissues from a type Ldh,-S coalfish (Syner & Goodman gel). The tissues were: 1, retina; 2, red skeletal muscle; 3, liver; 4, spleen; 5, gill; 6, brain ; 7, erythrocytes; 8, gas gland.
with their presence in retina, spleen and erythrocytes. The red skeletal muscle, which lies under the lateral line system, differs from white skeletal muscle and resembles the heart in that the H, isoenzyme is stronger than K,. Red skeletal muscle and gill tissue also contain the minor isoenzyme which is present in heart muscle. The highly vascular gas gland of the swim bladder was expected to show some unique or unusual LDH zymogram because the production of gas by this tissue for hydrostatic purposes is thought to involve the secretion of lactic acid (Alexander, 1966); however, it showed only K, and H,. The polymorphism of the K polypeptide was apparent in every tissue. Testis was not investigated. Before it was found that Syner & Goodman gels give such a clear separation of K, from H,, most of the coalfish referred to in Table 1 had already been typed in Poulik gels. Figure 3 shows that in this gel the K, isoenzymes have mobilities similar to the H, isoenzyme; moreover, the K, homotetramers are accompanied by two satellite bands. Although the relative positions of the K, and H, isoenzymes are so different in the two kinds of gel the L, isoenzymes of liver, erythrocyte and retina correspond exactly in both gels, which confirms their identity. The identity of H, in different tissues was similarly confirmed. It is clearly helpful to examine complex mixtures of LDH isoenzymes in more than one buffer system, a point which will be reinforced in the following section on cod. A consistent but unexplained finding in the Poulik gels was the tendency of the H,L, isoenzyme in all three phenotypes to split into two bands, the slower one being the weaker. This effect was noticed with the corresponding cod
28
I. E.
LUSH
isoenzyme in both types of gel (see below). Syner & Goodman (1966) found similar satellite bands on the anodal side of the H,M, isoenzyme of the gelada baboon LDH.
F
FS
s
F
FS
s
F
FS
S
h
.,
-_
FIG. 3.
Diagram of LDH zymograms of heart, retina and white skeletal muscle from coalfish of the three Ldh, phenotypes (Poulik gel). Samples as in Fig. 1.
Cod Odense et al. (1966, 1969) h ave described an LDH polymorphism in samples of cod which were caught off the Atlantic coast of Canada. They found three common phenotypes which could be recognized by starch-gel electrophoresis of a variety of tissues including heart and retina. I have examined tissues from 184 cod caught within a distance of 40 miles of Aberdeen. Most of these were typed from the zymograms of retinal extracts in Poulik gels. The polymorphism described by Odense et al. (1966) was clearly recognizable, in fact the zymograms appeared to be very similar to theirs even although they used a different buffer system for electrophoresis. The correspondence was checked by comparing, in the same Poulik gel, extracts of retinas of each phenotype supplied by Dr. Odense with extracts of retinas of locally caught cod. The three phenotypes appeared to be the same in each population, confirming that we were dealing with the same polymorphism in Scottish cod as in Canadian cod. The LDH zymograms obtained in a Poulik gel with extracts of heart, retina and white skeletal muscle from cod of each phenotype are shown in Fig. 4. In muscle, the fast major isoenzyme predominates and showed no sign of genetic variation in forty individuals. It can be assumed to be a homotetramer of a K polypeptide determined by a locus homologous with the polymorphic Ldh, locus
COALFISH AND COD
LDH
VARIATION
29
of coalfish. The K, isoenzyme of cod, like that of coalfish in Poulik gels, is accompanied by satellite bands in front and behind. Farther back are traces of the genetically variable isoenzymes which form the predominant isoenzymes of heart. The three cod phenotypes are clearly due to variation of the H polypeptide and they will therefore be designated Ldhn-F, Ldhn-FS and Ldh,-S. The zymogram of the heart of the Ldhn-FS cod shows the five equally spaced bands which are characteristically found when two different polypeptides combine The heart zymograms of Ldhn-F and together at random to form tetramers. Ldhn-S cod have major isoenzymes which correspond, respectively, with the fastest and slowest isoenzymes of the heterozygote. The two corresponding alleles at the Ldh, locus will be referred to as Ldhg and Ldh$. The numbers of each phenotype found in a total of 184 cod caught between December 1966 and June 1968 are shown in Table 1. There is a slight but significant excess in the proportion of homozygotes compared with the Hardy-Weinberg expectations (x2 = 5.08, P
30
I. E. LUSH
in a Syner & Goodman gel. The zymogram of muscle shows K, and a small amount of the polymorphic H,, although the H, isoenzymes are hardly visible in the heterozygote. In the retinas the K, isoenzyme falls in the same place as in muscle, although it happens to coincide with other retinal isoenzymes. The retinal array of isoenzymes formed by combinations of L and the polymorphic H polypeptides is less clearly defined than in the Poulik gel but can nevertheless be seen to give the same phenotypes, F
FS
s
F
FS
S
F
FS
FIG. 5. Diagram of zymograms of heart, retina and white cod of each of the Ldh, phenotypes (Syner & Goodman gel).
S
skeletal muscle from Samples as in Fig. 4.
In Fig. 5 the heart zymograms of the three phenotypes show the expected H, patterns and the isoenzyme K, is also present in each. However, the bands enclosed in square brackets in Fig. 5 have no counterparts in the Pouiik zymogram and as yet they have no firm explanation in terms of polypeptide composition. The pattern of these anomalous isoenzymes in the three phenotypes suggests that they are heterotetramers made up of two H polypeptides combined with two polypeptides of a much slower mobility. We may symbolize these isoenzymes by H,Q,. In fact in a number of experiments in which the length of the run or the composition of the Syner & Goodman buffer were altered slightly it was found that the H,Q, isoenzymes always correspond exactly with the retinal H,L, isoenzymes of the corresponding phenotype. However, if the anomalous isoenzymes really are H,L, why do they not appear in heart zymograms in Poulik
COALFISH AND COD
LDH
VARIATION
31
gels ? It would also be of interest to know if they are in some way comparable to the anomalous (square bracketed) isoenzymes of coalfish shown in Fig. 1, although from the genetical evidence this seems unlikely. The distribution of LDH isoenzymes in other cod tissues broadly resembles that found in coalfish. When the LDH isoenzymes of a coalfish and an Ldhn-F cod were compared in the same gels, it was found that the mobilities of the five isoenzymes of the HL series were the same in the two species. This was true for each of the two buffer systems. However Dr. P. Odense (personal communication) has evidence which may show that the L4 isoenzymes of cod liver and retina are electrophoretically separable under some conditions. DISCUSSION
One conclusion from this survey of cod and coalfish LDH is that both species have three principal LDH loci active in their somatic tissues. The comparable loci in the two species are so similar in their tissue-specific expression that they can be assumed to be homologous. In terms of the H, L and K nomenclature adopted above, one finds that the H and L polypeptides combine together to form heterotetramers but there is no clear evidence of combination between K and the other two. In cod the H locus is polymorphic while in coalfish the K locus is polymorphic. Another conclusion is that, because of the unpredictable occurrence of tissuespecificity and genetic variation, it is unwise to arrive at an estimate of the number of LDH loci in the genome of a fish species without a survey of different tissues and a number of individuals. The comparison of zymograms obtained with different buffer systems will add conviction to the estimate. In both cod and coalfish the LDH zymogram of red skeletal muscle resembles that of the heart and differs from that of the white skeletal muscle in the predominance of H, over K,. Fish red skeletal muscle is used for slow and steady cruising over long periods, while the white muscle comes into use for rapid movements of short duration (Bone, 1966). Th e energy metabolism of red muscle is mainly aerobic while white muscle is rapidly fatigued, presumably by the accumulation of an oxygen debt. This is consistent with the finding that red muscle resembles heart muscle in its LDH zymogram. With regard to the Ldh gene frequencies, the data have not produced any surprises. The frequencies in the three coalfish populations are obviously very similar, and the gene frequency of the local Aberdeen cod population is almost exactly the same as in cod off the Atlantic coast of Canada which Odense et al. (1966) found to have an Ldhg frequency between 0.35 and O-39. The large differences between the haemoglobin gene frequencies in Scottish and Canadian cod (O-65 and O-04, respectively for the H6P allele), as well as other biological considerations, shows that the two populations are entirely separate (Sick, 1965). It would be of interest to see if the cod LDH polymorphism, unlike the hemoglobin polymorphism, is maintained at the same gene frequency throughout the range of the species. 2
I. E.
32
LUSH
Acknowledgements-My thanks are due to Dr. P. T. Grant for his help and encouragement, and to Mr. D. Knox for technical assistance.
REFERENCES ALEXANDERR. McN. (1966) Physical aspects of swimbladder function. BioE. Rev. 41, 141-176. BONE Q. (1966) On the function of the two types of myotomal muscle fibre in elasmobranch fish. J. mar. biol. Ass. U.K. 46, 321-349. DAUGHERTYW. (1965) Analysis of the apparently single form of lactate dehydrogenase (LDH) found in the flatfish, Paralichthys dentatus. Am. Zool. 5, 20.5. FRITZ P. J. & JACOBSONK. B. (1965) Multiple molecular forms of lactate dehydrogenase. Biochemistry 4, 282-289. KAPLAN N. 0. & GOODFRIENDT. L. (1964) Role of the two types of lactic dehydrogenase. Adv. Enz. Regul. 2, 203-212. LUSH I. E., COWEY C. B. & KNOX D. (1969) The lactate dehydrogenase isoenzymes of twelve species of flatfish (Heterosomata). J. exp. Zool. (In press.) MARKERT C. L. & FAULHABERI. (1965) Lactate dehydrogenase isozyme patterns of fish. J. exp. Zool. 159, 319-332. MORRISON W. J. & WRIGHT J. E. (1966) G enetic analysis of three lactate dehydrogenase isozyme systems in trout: evidence for linkage of genes coding subunits A and B. J. exp. 2001. 163, 259-270. ODENSE P. H., LEUNG T. C. & ALLEN T. M. (1966) An electrophoretic study of tissue proteins and enzymes of four Canadian cod populations. Cons. Perm. Int. Explor. Mer. C.M. No. G, 14, 1. ODENSEP. H., LEUNG T. C. & ALLEN T. M. (1969) Multiple forms of lactic dehydrogenase in the cod. Biochem. Genetics 3, 317-324. PESCE A., FONDY T. P., STOLZENBACHF., CASTILLO F. & KAPLAN N. 0. (1967) The comparative enzymology of lactic dehydrogenase-III. Properties of the H, and M, enzymes from a number of vertebrates. J. biol. Chem. 242, 2151-2167. POULIK M. D. (1957) Starch gel electrophoresis in a discontinuous system of buffers. Nature, Lond. 180, 1477. SHAW C. R. & BARTOE. (1963) Genetic evidence for the subunit structure of lactate dehydrogenase isozymes. Proc. natn Acad. Sci. U.S.A. 50, 211-214. SICK K. (1965) Haemoglobin polymorphism of cod in the North Sea and the South Atlantic Ocean. Hereditas 54, 49-73. STAMBAUGHR. & BUCKLEY J. (1967) The enzymic and molecular nature of the lactic dehydrogenase subbands and X4 isoenzyme. r. biol. Chem. 242, 40534059. SYNER F. N. & GOODMANM. (1966) Polymorphism of lactate dehydrogenase in gelada baboons. Science 151, 206-208. Key
Word Index-Coalfish
; cod ; isoenzyme ; lactate dehydrogenase;
polymorphism.