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Electrophoretic patterns of malate and lactate dehydrogenases in a freshwater catfish
BiochemicalSystemaEcsand Ecology,Vol. 21, No. 2, pp. 209-214, 1993. Printed in GreatBritain.
0305-1978/93 $6.00+0.00 © 1993PergamonPressLtd.
Electrophoretic Patterns of Malate and Lactate Dehydrogenases in a Freshwater Catfish SHRI P. SHUKLA and GHANASHYAM TRIPATHI Department of Zoology, Banaras Hindu University, Varanasi 221005, India
Key Word Index--Catfish; Clariidae; Cypriniformes; species-specificity.
polyacrylamide gel electrophoresis; isoenzymes;
Abstract--The electrophoretic pattern of cytoplasmic (cMDH) and mitochondrial (mMDH) malate dehydrogenase and lactate dehydrogenase (LDH) was studied in the liver and the skeletal muscle of the freshwater catfish, C/arias batrachus. The cMDH and mMDH in both tissues showed the presence of two distinct isoforms, A and B, in which A form of cMDH was predominant in the liver. The existence of LDH in two isoenzymic forms, A and B, in the skeletal muscle and only A form in the liver indicated tissue-specific expression of LDH isoenzymes.
Introduction
Malate dehydrogenase (MDH, I-malate, NAD+-oxidoreductase, EC 1.1.1.37) has two isoenzymic forms: cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH). These isoenzymes not only differ in their kinetic properties but also differ in their electrophoretic properties (Bailey eta/., 1969; Bailey et a/., 1970; Durham eta/., 1980; Schwantes and Schwantes, 1982a, b; Tikhomirova, 1984). Both isoenzymes in fish exist as dimers composed of A and B subunits, that are under the control of two separate genes (Bailey eta/., 1969; DeLuca et al., 1983; Frankel and Wilson, 1984). The cMDH from most fish appears as three sharp, equally spaced anodal bands, AA, AB and BB, where as the presence of one to three bands of mMDH has been reported in many fish (Whitt, 1970c; Clayton et al., 1973; Schwantes and Schwantes, 1982a,b; DeLuca et al., 1983; Frankel and Wilson, 1984). The number of isoenzymes of lactate dehydrogenase (LDH, I-lactate; NAD+-oxidoreductase, EC 1.1.1.27) in fish varies from one to 20 (Markert and Faulhaber, 1965; Whitt eta/., 1975; Frankel, 1983; Chatterjee and Jyotidhar, 1985; Kettler and Whitt, 1986; Tsoi and Lee, 1987). In addition to A and B types, the C type of LDH isoenzyme has also been reported in fish. It is expressed in heart, liver, gill, kidney and gonad of sturgeon (Markert, 1975). The C type LDH isoenzyme limits its expression to neural tissues of some teleosts such as eye and brain and termed as E4 (eye band) or retinal isoenzyme 1 (Lush eta/., 1969; Whitt, 1970a). It is also expressed in the liver of cod and carp and referred to as F4 (Sensabough and Kaplan, 1972; Shaklee et a/., 1973). However, this isoenzyme is not present in the liver, skeletal muscle and heart of many advanced teleosts (Chatterjee and Jyotidhar, 1985). The present study on electrophoretic pattern of MDH and LDH from the liver and the skeletal muscle of the freshwater catfish, C/arias batrachus, forms a supplement to existing information and offers correlation between isoenzymic patterns in other fish. Materials and Methods Fish. Freshwater catfish (C/arias batrachus) weighing 70-75 g and having a body length of 18-19 cm were collected during regressed phase (November to February) from local ponds around Varanasi, India. They were acclimatized to laboratory conditions in glass aquaria containing chlorine free tap water. Extraction of enzymes. The procedures adopted for subcellular fractionation and extraction of cMDH, mMDH
(Received 5 May 1992) 209
210
S. P. SHUKLA AND G. TRIPATHI
and LDH from the liver and the skeletal muscle were the same as described previously (Tripathi and Shukla, 1989). Gel electrophoresis. The polyacrylamide gel electrophoresis for enzymes was carried out according to the method of Dietz and Lubrano (1967). The slab gel acrylamide concentration was 8% for cMDH and mMDH and 6% for LDH. Specific units of each enzyme were loaded in separate lanes. The electrophoresis was carried out at 4°C and a current of 100 V was applied to the gel for 14 h. The multiple forms of MDH and LDH were detected by specific staining according to the method of Thorne et al., (1963) and Dietz and Lubrano (1967). The gels were stained under conditions of complete darkness at an incubation temperature of 50°C.
Results and Discussion Electrophoretic patterns of cMDH, mMDH and LDH in the liver are shown in Fig. 1. The cMDH and mMDH possess two distinct isoforms, A and B, while LDH exhibits one enzymic form which corresponds to A form. Each of these enzymes in the skeletal muscle exists as two separate isoforms (Fig. 2). The presence of two isoforms, A and B, of cMDH in the liver and the skeletal muscle of C. batrachus (Figs 1, 2) is in contrast to cMDH from most fish having three distinct forms (Bailey et al., 1969; Bailey et al., 1970; Whitt et al., 1973; Schwantes and Schwantes, 1982b; DeLuca et al., 1983; Frankel and Wilson, 1984; Tikhomirova, 1984), six to eight bands in the saury, Cololabis saira (Numachi, 1970b), one band in some primitive fish like Acipencer oxyrhynchus and Amia calva and also in the catfish, Ictalurus nebulosus (Schwantes and Schwantes, 1982a). The predominance of A isoform of cMDH in the liver of C. batrachus is in agreement with the view of Fisher et al., (1980) who proposed that A form often predominates in the liver of most fish. Likewise, the occurrence of two isoforms of mMDH in the liver and the skeletal muscle (Figs 1, 2) is again in contrast to the presence of three bands in many fish (Kitto and Lewis, 1967; Whitt, 1970c; Clayton et al., 1973; Schwantes and Schwantes, 1982a), and one band in king salmon (Bailey et al., 1970) as well as in the tropical fish, Astyanax fasciatus (DeLuca et al., 1983). The electrophoretic pattern further reveals that the isoforms of cMDH and mMDH in the skeletal muscle may have identical charges, hence, correspond in their mobility. However, in the liver, the B isoform of cMDH bears more negative charges than the A isoform as a result of which the latter remains confined towards the cathodal end. The differences in the mobility between the isoforms of mMDH and cMDH may also be due to differences in the negative charges they possess. These results indicate that there are two types of cMDH and two types of mMDH in the liver and the skeletal muscle of this catfish and they differ in their electrophoretic mobility. The occurrence of a single band of LDH in the liver of C. batrachus (Fig. 1) is similar to the observations in the liver of some fish (Markert etal., 1975; Whitt, 1984) but differs by the presence of more than one band in the liver of other fish (Hochachka, 1969; Shaklee and Whitt, 1981; Frankel, 1983; Kettler and Whitt, 1986). Similarly, the presence of two isoenzymes in the skeletal muscle LDH (Fig. 2) is in agreement with the presence of two bands in the skeletal muscle of Fundulus heteroclitus, Scomber scombrus, Nocomis biguttatus, Galeus melastomus, Urophycis chuss and Coryphaenoides rupestris (Whitt et al., 1975; Totland et al., 1978; Shaklee and Whitt, 1981). It differs with the presence of a single LDH band as well as more than two bands in the skeletal muscle of various groups of fish (Markert et al., 1975; Whitt et al., 1975; Whitt, 1981; Whitt, 1984; Rehse and Davidson, 1986; Whitt, 1986). The third LDH isoenzyme, LDH-C4, is not expressed in the liver and the skeletal muscle of this catfish. Similar observations have already been reported in five species of Channa (Chatterjee and Jyotidhar, 1985). It has been suggested that the expression of a C type of LDH isoenzyme is restricted to neural tissues such as eye and brain in advanced teleosts. The existence of one LDH band in the liver and two bands in the skeletal muscle of C. batrachus provides evidence for differential expression of LDH in two different metabolic tissues. It indicates tissue-specific adaptation of LDH isoenzymes for aerobic and anaerobic glycolysis in the liver and skeletal muscle respectively.
211
m MDH
cMDH
LDH
ii ¸
A
B
A
A
B
1 4-
Liver FIG. 1. POLYACRYLAMIDE GEL ELECTROPHORESlS OF cMDH, mMDH AND LDH FROM THE LIVER OF C. BATRACHUS
212
cMDH
LDH
m MDH
i~::
A B
A B
A
B
~i i~!ii¸
l +
Skeletal muscle FIG. 2. POLYACRYLAMIDE GEL ELECTROPHORESIS OF cMDH, mMDH AND LDH FROM THE SKELETAL MUSCLE OF C BATP.ACHUS
MALATEAND LACTATEDEHYDROGENASESIN FRESHWATERCATFISH
213
Acknowledffements--This work was supported by a research grant (J-12017/28) to S. P. S. from the Ministry of Environment and Forests, Government of India, New Delhi.
References Bailey, G. S., Cocks, G. T. and Wilson, A. C. (1969) Gene duplication in fishes: Malate dehydrogenase of salmon and trout. Biochern. Biophys. Res. Comrnun. 34, 605-612. Bailey, G. S., Wilson, A. C., Halver, J. E. and Johnson, C. L. (1970) Multiple forms of supernatant malate dehydrogenase in salminid fishes. J. Biol. Chem. 245, 5927-5940. Chatterjee, K. and Jyotidhar, N. (1985) Lactate dehydrogenase isoenzymes in the genus Channa: Speciesspecific patterns. Biochem. Syst. Ecol. 13, 467-470. Clayton, J. W., Harris, R. E. K. and Tretiak, D. N. (1973) Identification of supernatant and mitochondrial isoenzymes of malate dehydrogenase on electrophorograms applied to the taxonomic discrimination of walleye (SEzolection vitreum vib'eum), sanger (S. canadense) and suspected interspecific hybrid fishes. J. Fish Res. Bd. Can. 30, 927-938. DeLuca, P. H., Schwantes, M. L. B. and Schwantes, A. R. (1983) Adaptative features of ectothermic enzymes-IV. Studies on malate dehydrogenase of Astyanax fasciatus (Characidae) from Lobo reservoir (S&o Carlos, S~o Paulo, Brasil). Comp. Biochem. Physiol. 74B, 315-324. Dietz, A. A. and Lubrano, T. (1967) Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Analyt. Biochem. 20, 246-257. Dunham, R. A., Philipp, D. P. and Whitt, G. S. (1980) Levels of duplicate gene expression in armoured catfishes. J. Hered. 71, 248-252. Fisher, S. E., Shaklee, J. B., Ferris, S. D. and Whitt, G. S. (1980) Evolution of five multilocus isozyme systems in the chordates. Genetics 52/53, 73-85. Frankel, J. S. (1983) Lactate dehydrogenase tissue specificity and characterization in the teleost genus Barbus. Comp. Biochem. Physiol. 76B, 103-106. Frankel, J. S. and Wilson, R. V. (1984) Comparison of the spatial and temporal expression of supernatant malate dehydrogenase in Barbus hybrids (Cypriniformes, Teleostei). Comp. Biochern. Physiol. 78B, 179-182. Hochachka, P. W. (1989) Intermediary metabolism in fishes. In Fish Physiology (Hoar, W. S. and Randall, D. J., eds), Vol. I, pp. 351-389. Academic Press, New York. Kettler, M. K. and Whitt, G. S. (1986) An apparent progressive and recurrent evolutionary restriction in tissue expression of a gene, the lactate dehydrogenase--C gene, within a family of bony fish (Salmoniformes: Umbridae). J. Molec. Evol. 23, 95-107. Kitto, G. B. and Lewis, R. G. (1967) Purification and properties of tuna supernatant and mitochondrial malate dehydrogenases. Biochim. Biophys. Acta 138, 1-15. Lush, J. E., Cowey, C. B. and Knowx, D. (1989) The latate dehydrogenase isozymes of twelve species of flatfish (Heterosomata). J. Exp. Zoo/. 171, 105-118. Markert, C. L. and Faulhaber, I. (1965) Lactate dehydrogenase isozyme patterns of fish. J. Exp, Zoo/. 159, 319332. Markert, C. L., Shaklee, J. B. and Whitt, G. S. (1975) The evolution of a gene. Science 189, 102-114. Numachi, K. (1970b) Polymorphism of malate dehydrogenase and genetic structure of juvenile populations in saury Cololabis saira. Bull. Jap. Soc. Sc~ Fish. 36, 1235-1241. Rehse, P. H. and Davidson, W. S. (1986) Evolutionary relationship of a fish C type lactate dehydrogenase to other vertebrate lactate dehydrogenase isozymes. Can. J. Fish Aquat. ScL 43, 1045-1051. Schwantes, M. L. B. and Schwantes, A. R. (1982a) Adaptive features of ectothermic enzymes. I. Temperature effect on the malate dehydrogenase from a temperate fish, Leiostornus xanthurus. Cornp. Biochem. Physiol. 72B, 49-58. Schwantes, M. L. B. and Schwantes, A. R. (1982b) Adaptive features of ectothermic enzymes. I1. The effect of acclimation temperature on the malate dehydrogenase of the spot, Leiostomus xanthurus. Comp. Biochem. Physiol. 72B, 59-64. Sensabaugh, G. F. and Kaplan, N. O. (1972) A lactate dehydrogenase specific to the liver of gadoid fish. J. Biol. Chem. 247, 585-593. Shaklee, J. B., Kepes, K. L. and Whitt, G. S. (1973) Specialized lactate dehydrogenase isozymes: A molecular and genetic basis for the unique eye and liver LDHs of teleost fish. J. Exp. Zoo/. 185, 217-240. Shaklee, J. B. and Whitt, G. S. (1981) Lactate dehydrogenase isozymes of gadiform fishes: Divergent patterns of gene expression indicate a heterogenous taxon. Copeia 3, 563-578. Thorne, C. J. R., Grossnen, L. and Kaplan, N. O. (1963) Starch gel electrophoresis of rnalate dehydrogenase. Biochim. Biophys. Acta 73, 193-203. Tikhomirova, G. 1". (1984) Multiple forms of malate dehydrogenase and their inheritance in carp (Cyprinus carpio L.) Genetika 20, 817-820. Totland, G. K., Kryvi, H. and Slinde, E. (1978) LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax. J. Fish, Biol. 12, 45-50. Tripathi, G. and Shukla, S. P. (1989) Induction of cytoplasmic and mitochondrial enzymes by thryoid hormone in a freshwater catfish. Biochem. Int. 19, 045-949.
214
S.P. SHUKLAAND G. TRIPATHI
Tsoi, S. C. M. and Lee, S. C. (1987) An electrophoretic investigation of tissue-specific isozyme of lactate dehydrogenase in some holocentrid fishes from Taiwan. Bull. Inst. Zoo/. Acad. Sinica 26, 151-156. Whitt, G. S. (1970a) Developmental genetics of the lactate dehydrogenase isozymes of fish. J. Exp. Zoo/. 175, 1-36. Whitt, G. S. (1970c) Genetic variation of supernatant and mitochondrial malate dehydrogenase isozymes in the teleost Fundulus heteroclitus. Experientia 26, 734-736. Whitt, G. S., Childers, W. F., Tranquilli, J. and Champion, M. (1973) Extensive heterozygosity at three enzyme loci in hybrid sunfish populations. Biochem. Genet. 8, 55-71. Whitt, G. S., Shaklee, J. B. and Markert, C. L. (1975) Evolution of the lactate dehydrogenase isozymes of fishes. In Isozymes (Markert, C. L., ed.), Vol. IV, pp. 381-399. Academic Press, New York. Whitt, G. S. (19~4) Genetic, developmental and evolutionary aspects of the lactate dehydrogenase isozyme system. Cell Biochem. Funct. 2, 134-139.
0305-1978/93 $6.00+0.00 © 1993PergamonPressLtd.
Electrophoretic Patterns of Malate and Lactate Dehydrogenases in a Freshwater Catfish SHRI P. SHUKLA and GHANASHYAM TRIPATHI Department of Zoology, Banaras Hindu University, Varanasi 221005, India
Key Word Index--Catfish; Clariidae; Cypriniformes; species-specificity.
polyacrylamide gel electrophoresis; isoenzymes;
Abstract--The electrophoretic pattern of cytoplasmic (cMDH) and mitochondrial (mMDH) malate dehydrogenase and lactate dehydrogenase (LDH) was studied in the liver and the skeletal muscle of the freshwater catfish, C/arias batrachus. The cMDH and mMDH in both tissues showed the presence of two distinct isoforms, A and B, in which A form of cMDH was predominant in the liver. The existence of LDH in two isoenzymic forms, A and B, in the skeletal muscle and only A form in the liver indicated tissue-specific expression of LDH isoenzymes.
Introduction
Malate dehydrogenase (MDH, I-malate, NAD+-oxidoreductase, EC 1.1.1.37) has two isoenzymic forms: cytoplasmic malate dehydrogenase (cMDH) and mitochondrial malate dehydrogenase (mMDH). These isoenzymes not only differ in their kinetic properties but also differ in their electrophoretic properties (Bailey eta/., 1969; Bailey et a/., 1970; Durham eta/., 1980; Schwantes and Schwantes, 1982a, b; Tikhomirova, 1984). Both isoenzymes in fish exist as dimers composed of A and B subunits, that are under the control of two separate genes (Bailey eta/., 1969; DeLuca et al., 1983; Frankel and Wilson, 1984). The cMDH from most fish appears as three sharp, equally spaced anodal bands, AA, AB and BB, where as the presence of one to three bands of mMDH has been reported in many fish (Whitt, 1970c; Clayton et al., 1973; Schwantes and Schwantes, 1982a,b; DeLuca et al., 1983; Frankel and Wilson, 1984). The number of isoenzymes of lactate dehydrogenase (LDH, I-lactate; NAD+-oxidoreductase, EC 1.1.1.27) in fish varies from one to 20 (Markert and Faulhaber, 1965; Whitt eta/., 1975; Frankel, 1983; Chatterjee and Jyotidhar, 1985; Kettler and Whitt, 1986; Tsoi and Lee, 1987). In addition to A and B types, the C type of LDH isoenzyme has also been reported in fish. It is expressed in heart, liver, gill, kidney and gonad of sturgeon (Markert, 1975). The C type LDH isoenzyme limits its expression to neural tissues of some teleosts such as eye and brain and termed as E4 (eye band) or retinal isoenzyme 1 (Lush eta/., 1969; Whitt, 1970a). It is also expressed in the liver of cod and carp and referred to as F4 (Sensabough and Kaplan, 1972; Shaklee et a/., 1973). However, this isoenzyme is not present in the liver, skeletal muscle and heart of many advanced teleosts (Chatterjee and Jyotidhar, 1985). The present study on electrophoretic pattern of MDH and LDH from the liver and the skeletal muscle of the freshwater catfish, C/arias batrachus, forms a supplement to existing information and offers correlation between isoenzymic patterns in other fish. Materials and Methods Fish. Freshwater catfish (C/arias batrachus) weighing 70-75 g and having a body length of 18-19 cm were collected during regressed phase (November to February) from local ponds around Varanasi, India. They were acclimatized to laboratory conditions in glass aquaria containing chlorine free tap water. Extraction of enzymes. The procedures adopted for subcellular fractionation and extraction of cMDH, mMDH
(Received 5 May 1992) 209
210
S. P. SHUKLA AND G. TRIPATHI
and LDH from the liver and the skeletal muscle were the same as described previously (Tripathi and Shukla, 1989). Gel electrophoresis. The polyacrylamide gel electrophoresis for enzymes was carried out according to the method of Dietz and Lubrano (1967). The slab gel acrylamide concentration was 8% for cMDH and mMDH and 6% for LDH. Specific units of each enzyme were loaded in separate lanes. The electrophoresis was carried out at 4°C and a current of 100 V was applied to the gel for 14 h. The multiple forms of MDH and LDH were detected by specific staining according to the method of Thorne et al., (1963) and Dietz and Lubrano (1967). The gels were stained under conditions of complete darkness at an incubation temperature of 50°C.
Results and Discussion Electrophoretic patterns of cMDH, mMDH and LDH in the liver are shown in Fig. 1. The cMDH and mMDH possess two distinct isoforms, A and B, while LDH exhibits one enzymic form which corresponds to A form. Each of these enzymes in the skeletal muscle exists as two separate isoforms (Fig. 2). The presence of two isoforms, A and B, of cMDH in the liver and the skeletal muscle of C. batrachus (Figs 1, 2) is in contrast to cMDH from most fish having three distinct forms (Bailey et al., 1969; Bailey et al., 1970; Whitt et al., 1973; Schwantes and Schwantes, 1982b; DeLuca et al., 1983; Frankel and Wilson, 1984; Tikhomirova, 1984), six to eight bands in the saury, Cololabis saira (Numachi, 1970b), one band in some primitive fish like Acipencer oxyrhynchus and Amia calva and also in the catfish, Ictalurus nebulosus (Schwantes and Schwantes, 1982a). The predominance of A isoform of cMDH in the liver of C. batrachus is in agreement with the view of Fisher et al., (1980) who proposed that A form often predominates in the liver of most fish. Likewise, the occurrence of two isoforms of mMDH in the liver and the skeletal muscle (Figs 1, 2) is again in contrast to the presence of three bands in many fish (Kitto and Lewis, 1967; Whitt, 1970c; Clayton et al., 1973; Schwantes and Schwantes, 1982a), and one band in king salmon (Bailey et al., 1970) as well as in the tropical fish, Astyanax fasciatus (DeLuca et al., 1983). The electrophoretic pattern further reveals that the isoforms of cMDH and mMDH in the skeletal muscle may have identical charges, hence, correspond in their mobility. However, in the liver, the B isoform of cMDH bears more negative charges than the A isoform as a result of which the latter remains confined towards the cathodal end. The differences in the mobility between the isoforms of mMDH and cMDH may also be due to differences in the negative charges they possess. These results indicate that there are two types of cMDH and two types of mMDH in the liver and the skeletal muscle of this catfish and they differ in their electrophoretic mobility. The occurrence of a single band of LDH in the liver of C. batrachus (Fig. 1) is similar to the observations in the liver of some fish (Markert etal., 1975; Whitt, 1984) but differs by the presence of more than one band in the liver of other fish (Hochachka, 1969; Shaklee and Whitt, 1981; Frankel, 1983; Kettler and Whitt, 1986). Similarly, the presence of two isoenzymes in the skeletal muscle LDH (Fig. 2) is in agreement with the presence of two bands in the skeletal muscle of Fundulus heteroclitus, Scomber scombrus, Nocomis biguttatus, Galeus melastomus, Urophycis chuss and Coryphaenoides rupestris (Whitt et al., 1975; Totland et al., 1978; Shaklee and Whitt, 1981). It differs with the presence of a single LDH band as well as more than two bands in the skeletal muscle of various groups of fish (Markert et al., 1975; Whitt et al., 1975; Whitt, 1981; Whitt, 1984; Rehse and Davidson, 1986; Whitt, 1986). The third LDH isoenzyme, LDH-C4, is not expressed in the liver and the skeletal muscle of this catfish. Similar observations have already been reported in five species of Channa (Chatterjee and Jyotidhar, 1985). It has been suggested that the expression of a C type of LDH isoenzyme is restricted to neural tissues such as eye and brain in advanced teleosts. The existence of one LDH band in the liver and two bands in the skeletal muscle of C. batrachus provides evidence for differential expression of LDH in two different metabolic tissues. It indicates tissue-specific adaptation of LDH isoenzymes for aerobic and anaerobic glycolysis in the liver and skeletal muscle respectively.
211
m MDH
cMDH
LDH
ii ¸
A
B
A
A
B
1 4-
Liver FIG. 1. POLYACRYLAMIDE GEL ELECTROPHORESlS OF cMDH, mMDH AND LDH FROM THE LIVER OF C. BATRACHUS
212
cMDH
LDH
m MDH
i~::
A B
A B
A
B
~i i~!ii¸
l +
Skeletal muscle FIG. 2. POLYACRYLAMIDE GEL ELECTROPHORESIS OF cMDH, mMDH AND LDH FROM THE SKELETAL MUSCLE OF C BATP.ACHUS
MALATEAND LACTATEDEHYDROGENASESIN FRESHWATERCATFISH
213
Acknowledffements--This work was supported by a research grant (J-12017/28) to S. P. S. from the Ministry of Environment and Forests, Government of India, New Delhi.
References Bailey, G. S., Cocks, G. T. and Wilson, A. C. (1969) Gene duplication in fishes: Malate dehydrogenase of salmon and trout. Biochern. Biophys. Res. Comrnun. 34, 605-612. Bailey, G. S., Wilson, A. C., Halver, J. E. and Johnson, C. L. (1970) Multiple forms of supernatant malate dehydrogenase in salminid fishes. J. Biol. Chem. 245, 5927-5940. Chatterjee, K. and Jyotidhar, N. (1985) Lactate dehydrogenase isoenzymes in the genus Channa: Speciesspecific patterns. Biochem. Syst. Ecol. 13, 467-470. Clayton, J. W., Harris, R. E. K. and Tretiak, D. N. (1973) Identification of supernatant and mitochondrial isoenzymes of malate dehydrogenase on electrophorograms applied to the taxonomic discrimination of walleye (SEzolection vitreum vib'eum), sanger (S. canadense) and suspected interspecific hybrid fishes. J. Fish Res. Bd. Can. 30, 927-938. DeLuca, P. H., Schwantes, M. L. B. and Schwantes, A. R. (1983) Adaptative features of ectothermic enzymes-IV. Studies on malate dehydrogenase of Astyanax fasciatus (Characidae) from Lobo reservoir (S&o Carlos, S~o Paulo, Brasil). Comp. Biochem. Physiol. 74B, 315-324. Dietz, A. A. and Lubrano, T. (1967) Separation and quantitation of lactic dehydrogenase isoenzymes by disc electrophoresis. Analyt. Biochem. 20, 246-257. Dunham, R. A., Philipp, D. P. and Whitt, G. S. (1980) Levels of duplicate gene expression in armoured catfishes. J. Hered. 71, 248-252. Fisher, S. E., Shaklee, J. B., Ferris, S. D. and Whitt, G. S. (1980) Evolution of five multilocus isozyme systems in the chordates. Genetics 52/53, 73-85. Frankel, J. S. (1983) Lactate dehydrogenase tissue specificity and characterization in the teleost genus Barbus. Comp. Biochem. Physiol. 76B, 103-106. Frankel, J. S. and Wilson, R. V. (1984) Comparison of the spatial and temporal expression of supernatant malate dehydrogenase in Barbus hybrids (Cypriniformes, Teleostei). Comp. Biochern. Physiol. 78B, 179-182. Hochachka, P. W. (1989) Intermediary metabolism in fishes. In Fish Physiology (Hoar, W. S. and Randall, D. J., eds), Vol. I, pp. 351-389. Academic Press, New York. Kettler, M. K. and Whitt, G. S. (1986) An apparent progressive and recurrent evolutionary restriction in tissue expression of a gene, the lactate dehydrogenase--C gene, within a family of bony fish (Salmoniformes: Umbridae). J. Molec. Evol. 23, 95-107. Kitto, G. B. and Lewis, R. G. (1967) Purification and properties of tuna supernatant and mitochondrial malate dehydrogenases. Biochim. Biophys. Acta 138, 1-15. Lush, J. E., Cowey, C. B. and Knowx, D. (1989) The latate dehydrogenase isozymes of twelve species of flatfish (Heterosomata). J. Exp. Zoo/. 171, 105-118. Markert, C. L. and Faulhaber, I. (1965) Lactate dehydrogenase isozyme patterns of fish. J. Exp, Zoo/. 159, 319332. Markert, C. L., Shaklee, J. B. and Whitt, G. S. (1975) The evolution of a gene. Science 189, 102-114. Numachi, K. (1970b) Polymorphism of malate dehydrogenase and genetic structure of juvenile populations in saury Cololabis saira. Bull. Jap. Soc. Sc~ Fish. 36, 1235-1241. Rehse, P. H. and Davidson, W. S. (1986) Evolutionary relationship of a fish C type lactate dehydrogenase to other vertebrate lactate dehydrogenase isozymes. Can. J. Fish Aquat. ScL 43, 1045-1051. Schwantes, M. L. B. and Schwantes, A. R. (1982a) Adaptive features of ectothermic enzymes. I. Temperature effect on the malate dehydrogenase from a temperate fish, Leiostornus xanthurus. Cornp. Biochem. Physiol. 72B, 49-58. Schwantes, M. L. B. and Schwantes, A. R. (1982b) Adaptive features of ectothermic enzymes. I1. The effect of acclimation temperature on the malate dehydrogenase of the spot, Leiostomus xanthurus. Comp. Biochem. Physiol. 72B, 59-64. Sensabaugh, G. F. and Kaplan, N. O. (1972) A lactate dehydrogenase specific to the liver of gadoid fish. J. Biol. Chem. 247, 585-593. Shaklee, J. B., Kepes, K. L. and Whitt, G. S. (1973) Specialized lactate dehydrogenase isozymes: A molecular and genetic basis for the unique eye and liver LDHs of teleost fish. J. Exp. Zoo/. 185, 217-240. Shaklee, J. B. and Whitt, G. S. (1981) Lactate dehydrogenase isozymes of gadiform fishes: Divergent patterns of gene expression indicate a heterogenous taxon. Copeia 3, 563-578. Thorne, C. J. R., Grossnen, L. and Kaplan, N. O. (1963) Starch gel electrophoresis of rnalate dehydrogenase. Biochim. Biophys. Acta 73, 193-203. Tikhomirova, G. 1". (1984) Multiple forms of malate dehydrogenase and their inheritance in carp (Cyprinus carpio L.) Genetika 20, 817-820. Totland, G. K., Kryvi, H. and Slinde, E. (1978) LDH isoenzymes in the axial muscle of the sharks Galeus melastomus and Etmopterus spinax. J. Fish, Biol. 12, 45-50. Tripathi, G. and Shukla, S. P. (1989) Induction of cytoplasmic and mitochondrial enzymes by thryoid hormone in a freshwater catfish. Biochem. Int. 19, 045-949.
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