Multiple forms of lactate dehydrogenase in the cod - Springer Link

Biochemical Genetics 3:317-334 (1969)

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L. P. H. Odense, 1 T. C. Leung, 1 T. M. Allen, 1 and E. Parker ~

Received 31 Oct. 1968--Final 18 Feb. 1969

Five types of subunits are postulated to explain the lactate dehydrogenase zymogram patterns of cod tissues. The subunits designated A, B, and C occur predominantly in white skeletal muscle, heart, and liver tissues, respectively. The D subunit is found almost exclusively in the retina. Activity of the E subunit is found in most tissues but does not appear to be predominant in any one tissue. The A subunits are most susceptible to heat treatment. The B subunits are the least reactive when AcPyAD, an N A D analogue, is used in place of NAD. Subbands may be produced in incubating heart or muscle extracts with pyloric caeca extract or with trypsin or chymotrypsin. Subbands normally appearing in tissues zymograms may represent partially synthesized or partially degraded active L D H tetramers normally present in a metabolizing tissue. In a sampling of three Canadian and one European cod populations, four forms of the heart subunit were found. They are designated B, B', B", and B'". Allele frequencies of the heart types for each population sample are compared. INTRODUCTION The isoenzyme patterns of fish lactate dehydrogenase (LDH) 2 are characteristic of the tissue and species. Markert and Faulhaber (1965) classified 30 species of fish on the basis of the number of L D H isoenzyme bands found in each species. While a two-subunit system accounts for most of the L D H patterns found in these fishes, Hochachka (1966) and Morrison and Wright (1966) found it necessary to postulate the presence of additional subunits to account for the complex L D H patterns of trout tissues. Genetic variants of the L D H subunits have also been found in fish tissues. Markert and Faulhaber (1965) found mutant forms of heart L D H subunits in silver hake (Merluccius bilinearis). Morrison and Wright (1966) described polymorphism of Fisheries Research Board of Canada, Halifax Laboratory, Halifax, Nova Scotia, Canada. 2 The abbreviations used are LDH, L-lactate: NAD oxidoreductase (E.C. 1.1.1.27); NAD, nicotinamide adenine dinucleotide; AcPyAD, 3-acetylpyridine analogue of NAD; EDTA, ethylene diamine tetraacetic acid; tris, tris (hydroxymethyl) aminomethane. 317

318

Odense, Leung, Allen, and Parker

the heart or B subunit of brook trout (Salvelinusfontinalis). Odense et al. (1966a, b) found both heart and muscle L D H subunit mutants in herring (Clupea harengus harengus) and heart subunit mutants in cod (Gadus morhua). More recently, Ohno et al. (1967) reported heart and muscle subunit mutants in hagfish (Eptatretus stoutii). In the present report the L D H isoenzyme system of the tissues of the cod is described. More than two subunit types must be postulated to describe the system. Mention must therefore be made of the terminology which will be used to describe the subunit formulation of the L D H tetramers. In a previous paper (Odense et al., 1966a), we used A and B to designate herring skeletal muscle and heart muscle subunits, respectively, in conformity with the convention established by Markert (1962). Additional subunit types have been described since, and no convention seems to exist for extending the nomenclature. Zinkham et al. (1963) described the new subunit in testes as the C subunit. Hochachka (1966) pointed out that as many as five subunits must be involved in the formation of the L D H isoenzymes of lower vertebrates. He designated the muscle and liver subunits of Salmonidae as A, B, C, D, and E. Morrison and Wright (1966) used C to indicate a retinal tissue subunit in Salmonidae. Thus the letters no longer indicate a particular tissue type. In the nomenclature used by Kaplan and his colleagues (Cahn et aL, 1962), the letter indicates the tissue where the particular L D H subunit predominantly occurs. However, we have found instances where the muscle type, designated M by Kaplan, shows more activity in the fish gas gland than it does in the muscle, while in some instances embryonic heart tissue may have a predominantly muscle type of subunit activity. It thus seems best to continue to use a less definitive but more applicable nomenclature. Therefore, we will use A, B, and C to designate the subunits predominantly present in white skeletal muscle, heart, and liver tissues, respectively. The letter D designates a subunit whose activity is almost exclusively restricted to retinal tissue. The subunit for a band of activity found in several tissue zymograms, but not especially typical of any one tissue, is termed E. The study of the genetic variation and distribution of heart subunit types previously reported (Odense et aL, 1966b) has been extended, and a new heart subunit mutant is described. The normal and mutant forms of the heart subunit are designated B, B', B", and B"', respectively. The B subunit and the mutant forms of the B subunit will be referred to collectively as the B-type subunit. MATERIALS AND METHODS Cod and tomcod (Microgadus tomcod L.) were caught near Halifax and kept at the laboratory in seawater aquaria at 2 C until required. For population studies cod were collected at Grande Rivi~re, Quebec, and the heart extracts were analyzed there. Other heart samples from cod collected at St. John's, Newfoundland, and Aberdeen, Scotland, were kept in ice and flown to Halifax for analysis. The fish were anesthetized with a solution of tricaine methane sulfonate 1 : 5000 in seawater before the tissues were excised. The tissues were homogenized for 1 rain in a Servall Omnimixer at 0 C with 2 vol of a 0.25 M sucrose, 0.001 M E D T A solution at p H 7.0. The extracts were centrifuged at 12,000 × g for 30 rain, and the supernatants were used directly for electrophoresis. All samples were taken from fresh

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

319

materials. Electrophoresis was usually performed the same day the sample was collected, although in a few cases the extracts or tissue samples were stored at 4 C for 2 days. Extracts of skeletal white muscle, heart muscle, intestine, liver, spleen, kidney, brain, gas gland, and gonad were examined. Retinas were similarly extracted and examined after they had been separated by dissection from the lens and humors. Plasma and hemolyzed red blood cells were also studied. The L D H isoenzymes were separated by vertical starch-gel electrophoresis based on the method of Smithies (1959). The tris-EDTA-borate buffer and the L D H staining solution used have been described (Odense et al., 1966a). In some cases a citrate buffer, p H 6.7, was used (Shows and Ruddle, 1968). Most samples were run for 18 hr at a constant 300 v and a current of 25 to 35 ma. For rapid typing of tissues, a 6-hr run at 400 v was sufficient. All procedures were carried out in the cold (0-4 C). In some instances the second half of the sliced gel was treated with an amido black solution to stain for total protein, was incubated with an NAD analogue (AcPyAD) in the LDHstaining solution, or was stained for "nothing dehydrogenase" by incubation in an LDH-staining solution lacking substrate (Shaw and Koen, 1965). For quantitative determinations of L D H activity of a tissue extract, the colorimetric method of Cabaud et al. (1958), described in Sigma 3 technical bulletin No. 500, was used. The pyruvic acid hydrazone formed by the unreacted pyruvate substrate was determined at 525 m# by the use of a Coleman Junior Spectrophotometer. The activity is reported in Berger-Broida L D H units per milliliter; one Berger-Broida unit is the amount of enzyme which will reduce 4.8 x 10 . 4 mole of pyruvate per minute at 25 C. The enzymes alpha chymotrypsin (three times recrystallized), trypsin (two times recrystallized), and pepsin (three times recrystallized) were purchased from the Nutritional Biochemicals Corporation. Tricaine methane sulfonate was obtained from Kent Chemicals Limited.

RESULTS AND DISCUSSION Thus far five different cod heart L D H patterns have been observed, representative of five different genotypes we have designated BB, BB', B'B', BB", and B B ' " . Cod are arbitrarily classified as L D H types I, II, III, IV, and V in the above order of genotypes. Tissues containing sufficient amounts of B-type subunits may be used to determine the cod L D H type. The tissue L D H patterns of the three common cod types (I, II, and III) illustrate this point (Figs. 1, 2, and 3). Type IV was observed in the heart pattern of a Halifax cod, while type V (Fig. 4) was found in the North Sea cod population sample. Other tissues of these two rare L D H types were not available. The five phenotype patterns found are represented in Fig. 5. Cod heart tissue produces A subunits, but they are apparent only as the two extra bands visible ahead of the B'4 band in the type II cod heart, representing A4 and A2B'2, a case of preferential association of homodimers (Odense et al., 1966a). Since the A 4 band coincides with the B 4 band in type I patterns run at pH 8.6, it is not possible to determine if the A subunits hydridize with B subunits. However, the type III 3 Sigma Chemical Co., St. Louis, Missouri.

320

Odense, Leung, Allen, and Parker

(-)12 ORIGIN---~

3

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(+1 Fig. 1. LDH zyrnogram of type I cod tissues. Tissues are (1) heart, (2) muscle, (3) liver, (4) spleen, (5) pyloric caeca, (6) eye, (7) brain, (8) gonad, (9) kidney, (10) gas gland. Electrophoresis conditions are the same for all figures: 18 hr at 300 v tris-EDTA-borate buffer, pH 8.6. heart pattern shows only heart bands, suggesting that A subunits hybridize with both B and B' and are thus diluted to the extent that only the predominant heart subunit patterns are visible. The p H of the electrophoretic run affected the mobilities of the A4 and B 4 bands differently. The Be band ran faster than A4 at p H 8.4 but slower at p H 9.4, while B'4 was always slower than A4 at these p H values. The potential for one kind of cell to produce all L D H subunit types is recognized

(-)

I

2

3

4

5

6

7

8 9

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(+) Fig. 2. LDH zymogram of type II cod tissues, same order in slots as Fig. 1.

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

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5

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7

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(+) Fig. 3. LDH zymogram of type III cod tissues same order in slots as Fig. 1. in c o d heart. In one instance a type I I I h e a r t sample clearly showed the retinal tissue p a t t e r n (Fig. 6a). W e f o u n d similar D subunit activity present in t o m c o d (Microgadus tomcod) h e a r t tissue (Fig. 6b). The b a n d s were weak a n d detectable only in fresh extracts.

(-)

15 14151211 I0 9 8 7 6 5 4 5 2 I

(+) Fig. 4. LDH zymogram of 15 North Sea cod heart extracts. Type Vcod heart pattern is seen in slot 10.

322

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Fig. 6. (a) LDH zymogram of a type IlI cod heart extract. Hybrid bands of D, B, and B' subunits are evident. (b) LDH zymogram of four tomcod heart extracts. D4, D3B, DzBz, DB3, B4, and As bands are visible.

(+) a

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A c t i v i t y o f the C s u b u n i t also occurs i n h e a r t tissue. T h e presence a n d e x t e n t o f the C4 b a n d activity in h e a r t p a t t e r n s is v a r i a b l e a n d suggests a c o r r e l a t i o n with a p h y s i o l o g i c a l c o n d i t i o n o f the cod r a t h e r t h a n a genetic trait (Fig. 7a). O t h e r tissues

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(+) Fig. 7a. LDH zymogram of four tissues of each of cod types I, II, and III. Slots contain from I to 12: liver II, I, III; heart II, I, III; muscle II, I, III; and eye II, I, III. Liver C4 activity is visible near origin and E, bands are present in liver, heart, and eye patterns at B'4 position.

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

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(+) Fig. 7b. Second half of gel in Fig. 7a stained for L D H activity with N A D analogue. Order of extracts is reverse of that in Fig. 7a. E4 activity is especially noticeable.

Table I. Frequency of Occurrence of Tetramer Combinations of D, B, and B' Subunits a Type I b tetramers D4 DaB

D2B2 DBa B4

x = 1

x = 2

x = 5

x = 10

1 4 6 4 1

1 8 24 24 16

1 20 150 375 600

1 40 600 4000 10000

1 2 2 1.5 3 1.5 0.5 1.5 1.5 0.5 0.06 0.25 0.375 0.25 0.06

1 4 4 6 12 6 3 9 9 3 1 4 6 4 1

1 10 10 37.5 75 37.5 46.9 140.6 140.6 46.9 37.5 150 225 150 37.5

Type IF tetramers

D4 D3B' D2B'2 DB'a B'4

Type III d tetramers

D4 DaB DaB'

D2B2 D2BB'

D2B'2 DB3 DB2B' DBB'2 DB'a B4 BaB'

BzB'2 BB'a

B'4

1 20 20 150 300 150 500 1500 1500 500 625 2500 3750 2500 625

a R a n d o m association of all subunits is assumed. b Type I cod: One D subunit synthesized for x subunits of B type. c Type II cod: One D subunit synthesized for x subunits of B' type. d Type III cod: One D subunit synthesized for x[2 subunits of B type and x/2 subunits of B' type.

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Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

327

show similar and parallel variability in C subunit activity. Since the C4 band persists after centrifuging the 0.25 M sucrose extract of heart tissue at 30,000 rpm, it cannot be attributed to bound or particulate L D H activity. Finally, a diffuse band of L D H activity found in several tissues was found in heart as well. Control gels incubated without substrate did not reveal any enzyme activity attributable to "nothing dehydrogenase" (Shaw and Koen, 1965), and the band has been tentatively designated E4. The retinal tissue patterns, like the heart patterns, reveal the presence of all types of subunits (Figs. 1, 2, 3, 7a, and 7b). The A4 band in retinal tissue is most apparent in type II patterns, but is obscured by the B4 band in type I and type III retinal tissue patterns. The D subunit hydridizes with both B and B' subunits. Since B type subunits are formed in excess of D subunits in retinal tissue, the resultant binomial distribution of L D H tetramers is skewed. Perhaps it is not always recognized how much the distribution is affected by the unequal production of the subunit types. In Table I the distribution of L D H types for varying ratios of D and the heart subunits B and B' has been calculated, assuming random association of subunits into tetramers. It is apparent that even a slight shift in the ratio of subunit types may decrease the amount of one or more tetramer types to below the level of detectability on the gel, and thus complicate further the interpretation of the zymograms. In this connection, a dilution series of a muscle extract containing 51,000 units of L D H activity per milliliter was run on a gel. After 5-hr incubation, activity could be detected in the sample in slot 14, containing only 6.3 units of L D H activity per milliliter (Fig. 8). The zymogram is thus capable of demonstrating widely varying ranges of L D H activity on the same gel. The D 4 band occurred in the same position as the C4 band when the t r i s - E D T A borate buffer was used, but these bands were separated by the citrate buffer (Fig. 9). The heart patter n containing the retinal tissue pattern shows B and D subunit hybridiz-

(-)

Fig. 9. LDH zymogram of cod: slot 1, liver; slot 2, blank; slot 3, retina. Citrate buffer, pH 6.7. B

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2

3

328

Odense, Leung, Allen, and Parker

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Fig. 10. LDH zymogram of four hemolysates of type IIl cod red blood cells. A and C hybridization is seen.

ation (Fig. 6), while heart patterns with strong C4 activity show no B and C or B' and C subunit hybridization (Fig. 7). Further evidence that these subunits differ is found in examining other tissue L D H patterns. In liver tissue L D H zymograms (Fig. 7) B type subunit bands are present, but again there is no evidence of B and C hybridization. However, hemolysates of red blood cells (Fig. 10) show C4 and A 4 activity and also bands indicative of C and A hybridization. Spleen, a vascular tissue, shows the same bands of C and A hybridization but no C and B hybridization, even though B subunits are present. The presence of the CaA, C/A2, and CA 3 bands in hemolysates and spleen is indicative of a more equal synthesis of A and C subunits in these tissues than in liver or muscle. Activity of the A subunit is present in most tissues, but especially skeletal muscle and gas gland. The subcellular localization of cod muscle L D H was attempted. A type I cod muscle homogenate was successively centrifuged to precipitate nuclei and cellular debris at 900 x g, mitochondria and lysosomes at 10,000 x g, and microsomes at 100,000 x g. A quantitative determination of the L D H activity in the supernatant following each centrifugation revealed no change in activity. It is suggested that the greater part of the L D H activity in cod muscle may be in a soluble form, unassociated with any cellular particulates. The heat stabilities of the various cod L D H isoenzymes were studied. Small portions of fresh extracts were heated in glass centrifuge tubes in a water bath at the desired temperature for 15 min. The extracts were then cooled quickly in an icewater bath and subjected to a quantitative determination of activity and to electrophoresis. The heart extract was least affected by temperature. Bands of L D H activity persisted in the heart extract after heating to 60 C (Fig. 11). Even after heating to 80 C the heart extract retained 2.4 x 104 L D H units of activity per gram of tissue. Both B and B' subunits showed this stability. The zymograms show that the muscle ex-

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

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15 14131211

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329

8 7 6 5 4 5 2

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(+) Fig. 11. LDH zymogram of heat-treated cod heart extracts. Slots 1, 4, 7, 10, and 13 contain type I extract heated to 30, 50, 60, 70, and 80 C, respectively. Slots 2, 5, 8, 11, and 14 contain type II extract similarly treated. Slots 3, 6, 9, 12, and 15 contain type III extract similarly treated. Activity is lost after heating to 70 C.

tracts lose their A4 b a n d activity after heating to 60 C, b u t the w e a k B'4 b a n d activity in muscle was n o t d e s t r o y e d until h e a t e d to 70 C (Fig. 12a). W h e n the second h a l f o f the muscle extract gel was stained for total p r o t e i n with a m i d o black, only one muscle a l b u m i n b a n d r e m a i n e d after heating to 60 C; this b a n d does n o t c o r r e s p o n d to the

(-)

5 4 5

2

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I

2

5

4

5

(+) a

b

Fig. 12. (a) LDH zymogram of heat-treated type IT cod muscle extract. Slots l, 2, 3, 4, and 5 contain muscle extract which was heated to 30, 40, 50, 60, and 70 C, respectively. Heart band B'~ persists after A4 activity is lost. (b) Second half of gel in Fig. 11a stained for protein with amido black. Muscle myogens are nearly all absent after heating to 60 C.

330

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(+) Fig. 13. LDH zymogram of heat-treated type II cod liver and eye extracts. Slots 1-5: eye extracts after heating to 30, 40, 50, 60, and 70 C, respectively. Slots 6-10: liver extracts similarly treated. Muscle band A4 is lost after heating to 60 C.

B' 4 L D H band (Fig. 12b). Retinal and liver tissue extracts continued to show activity until heated to 70 C (Fig. 13). The most anodic band in these extracts disappeared after heating to 60 C, further suggesting that this band is the A4 L D H band. Sometimes extra bands of L D H activity which did not correspond to the postulated tetramer combinations were observed in the gel patterns. A number of explanations may be given but have yet to be confirmed. Permutations of a tetramer may give rise to subbands (Markert and Faulhaber, 1965). A physiological adaptation may explain the occasional extra band seen in cod liver zymograms, similar to the changes observed by Hochachka (1965) in the liver L D H patterns of goldfish, Carassius carassius, adapted to different temperatures. Few subbands showed activity when AcPyAD was used in place of NAD. The A4, C4, and E4 bands retain activity, but B4, B'4 or tetramer combinations of B and B' are inactive in the presence of the analogue. The E 4 activity in the presence of the analogue (Fig. 7b) clearly distinguishes this band from the B type subunit tetramers and adds further evidence that the subunits of the E 4 band are a different type of subunit. Subbands are also present in all zymograms of pyloric caeca extracts (Figs. 1, 2, 3, 14a, 14b, and 14c). These bands reflect the cod B type subunit activity but stain less intensely, migrate further toward the anode, and are more numerous than the bands in the heart pattern or other tissues. Staining the other half of these gels with amido black reveals that the digestive enzymes have left no stainable protein and indeed have removed some of the background staining of the starch gel itself. The extra bands can be attributed to the partial digestion of the L D H by the enzymes of the pyloric caeca. This was demonstrated by incubating for various intervals at 40 C heart and muscle extracts of a type I cod together with equal portions of its pyloric caeca extract. Three bands of activity were found in both the pyloric caeca extract and the heart extract

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

(-)

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2

5

4

I

2

3

4

331

I

2

5

4

(+) a b c Fig. 14. (a) LDH zymogram of type I cod: spleen, pyloric caeca, eye, and brain extracts. (b) Type II cod, same tissues. (c) Type III cod, same tissues. Note subbanding of pyloric caeca tissue extracts.

mixed with the pyloric caeca extract. The mixture of muscle and pyloric caeca extract showed four bands. Only the pyloric caeca extract incubated for the longest period (2 hr at 40 C) showed no activity. This suggests that more than one amino acid or peptide can be removed from the L D H subunit without destroying its active site. Again, the second half of the gel revealed no amido black protein staining. To determine which digestive enzyme or enzymes might be responsible for the production of the subbands, portions of heart and muscle extracts were incubated with equal volumes of solutions of crystallized pepsin, trypsin, and chymotrypsin. The heart was extracted with a 0.25 M sucrose, 0.001 M E D T A solution, p H 7.0; 0.1 ml of l M CaC12 was added to the heart extract before mixing with the enzyme solutions. Muscle was extracted with 0.25 M sucrose and with 0.25 N sucrose brought to p H 8.0 with 0.01 M tris buffer. The former extract was combined with a 0.1% solution of pepsin in 0.05 M acetate buffer, p H 2.0, while the latter muscle extract was combined with 0.1% solutions of chymotrypsin or trypsin in the same tris buffer. For each enzyme plus extract solution, four conditions were observed: no heating, and heating at 40 C for 10 min, 30 min, and 2 hr. Controls with no enzymes added but diluted with equal volumes of the buffer were heated 10 min at 40 C. Following incubation, the samples were electrophoresed and stained for L D H and protein. The zymograms showed that trypsin, and to a lesser extent chymotrypsin, produced subbands migrating more rapidly toward the anode, thus resembling the effect of pyloric caeca extracts upon heart and muscle extracts. Pepsin had no apparent effect on muscle but completely inactivated the heart L D H , though the latter activity had already been greatly reduced by the effect o f p H , as was shown by the control. In the final experiment of this series,

332

Odense, Leung, Allen, and Parker

(-)

10 9 8 7 6 5 4 3 2

Fig. 15. L D H zymogram of type II cod muscle extract incubated with an equal amount of a 1 : 1 mixture of trypsin and chymotrypsin solution. Slots 1 and 10: control, no heating, and heating at 40 C for 10 rain. Slots 2-9: muscle extract with trypsin and chymotrypsin, no heating to heating at 40 C for 5, I0, 15, 30, 60, 120, and 240 rain, respectively.

1+)

a type II cod muscle extract was incubated at 40 C with an equal amount of a 1:1 mixture of the above-mentioned trypsin and chymotrypsin solutions. Again subbands appeared, while the main band decreased in intensity (Fig. 15). It is suggestive that the subbands formed coincide with the faint subbands frequently seen in muscle extracts. The results indicate that trypsin and chymotrypsin may be the enzymes in cod pyloric caeca which are responsible for the breakdown of L D H into active Table II. The Lactate Dehydrogenase (LDH) Genotypic Composition of One European and Three Canadian Cod Population Samples Compared to the Hardy-Weinberg Distribution L D H type Samplinglocality

I BB

II B'B'

III BB'

IV BB"

V BB"

Total

Obs. Exp.

43 36.7

21 14.8

34 46.5

0 0

0 0

98

Obs. Exp. Obs. Exp.

41 42.3 50 50.5

11 12.0 21 21.5

47 45.0 67 66.0

1 0.7 0 0

0 0 0 0

100

GrandeRivi~re, Quebec

Obs. Exp.

41 42.9

10 11.9

49 45.2

0 0

0 0

Aberdeen, Scotland

Obs. Exp.

28 26.7

23 21.0

43 46.8

0 0

1 0.5

St. John's, Newfoundland HNifax, Nova Scotia Catch l Catch2

138 100 95

Multiple Forms of Lactate Dehydrogenase in the Cod, Gadus morhua L.

333

Table Ill. Lactate Dehydrogenase Allele Frequencies in One European and

Three Canadian Cod Population Samples Allele Sampling locality B

B'

B"

B'"

St. John's, Newfoundland

0.61

0.39

0

0

Halifax, Nova Scotia

0.63

0.37

0.002

0

Grande Rivi6re, Quebec

0.65

0.35

0

0

Aberdeen, Scotland

0.53

0.47

0

0.005

fragments revealed as subbands. It also suggests an alternative explanation for subbanding--the presence of partially synthesized or partially degraded active L D H tetramers in an actively metabolizing tissue. The work on cod populations first reported by Odense et al. (1966b) now includes a study of the distribution of cod heart L D H phenotypes in two catches of cod from Halifax and one each from Grande Rivi6re, St. John's, and Aberdeen. The distribution of the five cod phenotypes and their agreement with the expected distribution calculated from the observed allele frequencies and based on the Hardy-Weinberg law is shown in Table II. The B, B', B", and B'" allele frequences in the four population samples are given in Table III. In three of the cod populations studied, the Halifax, Grande Rivi6re, and North Sea populations, the results agreed well with the expected Hardy-Weinberg distribution. This indicates that these three samples were representative of freely interbreeding populations that have attained genetic equilibrium. The excess of both kinds of homozygotes in the St. John's population may be explained if it is assumed that there are two noninterbreeding populations in the sampling area. Cod from the two sides of the Atlantic exhibited the same type of L D H polymorphism; however, the B allele frequency was considerably lower in the European cod population than in those from the three Atlantic regions. After enough sampling has been done to determine the allele frequency with some certainty, differences in allele frequencies as well as absolute differences in mutant forms may be used to distinguish populations and identify their origin. It is hoped that further sampling will help detect regions where cod populations are migrating and other regions where a single homogeneous population is present.

ACKNOWLEDGMENT We are greatly indebted to Mr. Noel Wilkins of the Marine Laboratory, Torry, Aberdeen, Scotland, for kindly providing us with the European cod population heart samples. We also wish to thank members of the Grande Rivi6re Station and St. John's

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