Comp. Biochem. Physiol. Vol. 102B, No. 4, pp. 673-677, 1992
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
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MINI REVIEW LACTATE DEHYDROGENASE IN TELEOSTS. THE ROLE OF LDH-C4 ISOZYME ZULEMA COPPES
Cfitedra de Bioquimica, Facultad de Quimica, General Flores 2124, Montevideo, Uruguay (Fax: 00598-2-941-906) (Received 23 September 1991)
Abstract--1. Lactate dehydrogenase (LDH) occupies an important position in cell metabolism. 2. Teleosts possess at least three genetic loci coding for lactate dehydrogenase subunits, Ldh-A, Ldh-B and Ldh-c. LDH exists in most tissues in several isozymic forms. 3. The isozyme LDH-C4 is synthesized predominantly in regions of the nervous system concerned with the eye.
INTRODUCTION
Several examples of homologous enzymes have been described in fishes: creatine kinase (CK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI) (Coppes et al., 1990). These multilocus isozyme systems were formed by relatively ancient gene duplications and are suitable candidates for the study of evolution of isozymic loci (Whitt, 1981 a). The genetic and molecular bases that give rise to their isozyme distribution are very well known. The number of isozymic loci present in a species is easily estimated by electrophoresis. Such loci diverged in most cases hundreds of millions of years ago. Thus, their isozymes differ considerably owing to innumerable amino acid substitutions. A corroboration of the number of loci present is obtained by the presence of genetic variants, and by the observation of the number and relative intensities of isozymes, as well as their physical and antigenic properties (Fisher et al., 1980; Whitt, 1981a). Lactate dehydrogenase is an isozyme system which, owing to its multigenic nature, is an interesting model for evolutionary studies, and from this aspect has been widely investigated (Wilson et al., 1964; Holmes, 1972; Whitt et al., 1973a,b, 1975; Markert et al., 1975; Fisher et al., 1980; Whitt, 1981b, 1987; Coppes, 1984; Coppes et al., 1990). In advanced fishes LDH is codified by three loci, Ldh-A, Ldh-B and Ldh-C. Ldh-C is restricted in its expression to neural tissues, eye and brain (in most advanced fishes) or liver (in some fishes). The present paper discusses the localization of the LDH-C4 isozyme in tissues of advanced fishes and tries to infer its function in retinal metabolism. LACTATE DEHYDROGENASE ISOZYMES
IN FISHES
Lactate dehydrogenase (lactate: N A D oxidoreductase, EC 1.1.1.27) with a tetrameric structure and a mol. wt of 14,000 (Darnall and Klotz, 1975)
catalyzes the interconversion of lactate and pyruvate. The reaction serves as an important source of oxidized coenzyme (nicotinamide adenine dinucleotide NAD +) during periods of transient anaerobiosis, or of a reduced form of such a coenzyme during aerobiosis. The existence of multiple loci encoding LDH has been well-documented for almost all vertebrate types (Coppes et al., 1990). In invertebrates, the enzymes catalyzing the pyruvate-lactate interconversion are not yet well characterized. In vertebrates, LDH is known to comprise a single homologous family (Markert et al., 1975). This family exists in a variety of isozymic forms, all related in that they catalyze the same chemical reaction, but all different from one another in molecular structure and commonly in genetic control. In vertebrates the polypeptide subunits of LDH are encoded in at least two genetic loci whose alleles are codominantly expressed (Markert, 1962; Shaw and Barto, 1963). According to Markert et al. (1975) only a single gene encoded the LDH polypeptide at the beginning of vertebrate evolution. The subunits encoded by this gene are able to polymerize to make a homotetramer with properties similar to those of the A4 isozyme commonly occurring in the skeletal muscle of all vertebrates. All contemporary vertebrates examined, save one, the Lampreia, have at least two genes coding for LDH polypeptides. The original A gene must have soon duplicated (Markert et al., 1975); later, these two A-like genes diverged by mutations to give rise to two distinctly different genes now designated A and B (Ohno, 1967; Markert et al., 1975; Whitt et al., 1975), which associate at random to yield binomial distributions of the five tetrameric isozymes (Appella and Markert, 1961; Markert et al., 1975). A third LDH locus has been found in many Orders of fish; the product of this third locus is referred to as the C4 isozyme (Shaklee et aL, 1973). This C 4 isozyme is like the B 4 isozyme in possessing a lower substrate concentration optimum and greater susceptibility to substrate inhibition than A4 isozyme in vitro 673
674
ZULEMA COPPES
(Shaklee et al., 1973). It is probable that these kinetic properties make the B 4 and C4 isozymes suited to tissues which are relatively more aerobic (Markert and Whitt, 1975). LDH-C ISOZYME IN FISHES
The Ldh-C locus present in most Actinopterygii (Coppes et al., 1990) was first observed in Chondrosteans (Fisher et aL, 1980). The Ldh-C locus probably arose from duplication of an Ldh-B locus (Whitt, 1969, 1970). In the primitive teleosts possessing a Ldh-C locus (Acipenseriformes, Amiiformes, Elopiformes, Anguilliformes and Osteoglossiformes) LDH-C4 isozyme is present in a variety of tissues (Markert et al., 1975; Whitt et al., 1975). LDH-C expression tends to be parallel to LDH-B in some primitive groups of teleosts (Fisher et al., 1980). In the advanced teleosts the structure and function of Ldh-C locus evolved in two different ways. In most Orders the gene encodes an anodic isozyme which predominates in "eye" and brain (Whitt, 1970, 1981a,b; Whitt et al., 1971, 1973b; Markert et al., 1975; Philipp and Whitt, 1977; Wiseman et al., 1978; Sassaman and Yoshiyama, 1979; Smith and Jamieson, 1978; Philipp et al., 1983; Paneppucci et al., 1984; Coppes et al., 1987; Basaglia, 1991). In some species of Cypriniformes and Gadiformes, the Ldh-C locus encodes a cathodic isozyme which predominates in liver (Markert and Faulhaber, 1965; Klose et al., 1969; Lush et al., 1969; Odense et al., 1969; Utter and Hodgins, 1969; Kepes and Whitt, 1972; Numachi, 1972; Sensabaugh and Kaplan, 1972; Whitt et al., 1973c; Wilson et al., 1973; Odense and Leung, 1975; Shaklee and Whitt, 1981; Coppes et al., 1990). The discovery of several fish species in which biochemically distinct eye- and liver-specific LDH isozymes are coexpressed, is discussed in the light of the currently accepted hypothesis that these two isozymes are encoded by a single locus (Ldh-C) which has undergone divergent tissue expression in several other major groups of teleosts (Holt and Leibel, 1987). The association between B and C subunits detected in neural tissues, commonly found in the literature, show that when a heteropolimer is present, it consists of B and C subunits (Morrison and Wright, 1966; Odense et al., 1969; Whitt, 1968, 1969, 1970, 1981b; Kepes and Whitt, 1972; Shaklee et al., 1973; Philipp and Whitt, 1977; Garlick and Terwilliger, 1978; Shaklee and Whitt, 1981; Coppes et al., 1987; Basaglia et al., 1990). Taking into account the physical, kinetic and immunochemical properties the C subunit in fishes, as well as in mammals and birds, is more similar to the B than to the A subunit. However, according to Whitt (1987), the data are not sufficient to decide whether Ldh-C gene is more closely related to A or to B. Shaklee et al. (1973) and Garlick and Terwilliger (1978) describe some species of fishes where associations of A and C subunits are detected. The results obtained by Coppes et al. (1987) suggest [through the method of serial dilutions of Klebe (1975)] that the C subunit of neural tissues is more similar to the B than to the A subunit.
DEVELOPMENTAL STUDIES
Tissue-specific isozyme patterns in the adult organism reflect developmental changes that occur in the control of gene expression. The presence of three homologous LDH loci in the teleost provides a system for examining this differential expression. The L D H - C 4 isozyme of teleosts is one example of an isozyme which is often restricted to a single cell type. In most species of teleosts, the Ldh-C gene appears to be expressed in the photoreceptor cells of the neural retina (Miller and Whitt, 1975). Thus, it has been possible to determine that the expression of the Ldh-C gene is tightly coordinated with the morphological and functional differentiation of the neural retina (Whitt, 1975). So, if LDH-C4 is important to retinal metabolism, its appearance during embryogenesis should occur concomitantly with morphological and functional differentiation of the retina. Thus, the timing of the synthesis of this retinal-specific isozyme with respect to the timing of the morphological differentiation was determined. Nakano and Whiteley (1965) reported that the "eye" band appears abruptly at hatching in the medaka (Oryzias latipes): they concluded that this was due to the conversion from anaerobic to an aerobic metabolism at hatching. Whitt (1970) observed the "eye" band at least 24 hr before the time of hatching in another teleost, Fundulus heteroelitus, and suggested that hatching was not the necessary stimulus to C-gene activation in this fish. In the trout, Hitzeroth et al. (1968) and Goldberg et al. (1969) showed that the retinal-specific LDH did not appear until a considerable time after hatching. Whitt (1968, 1970) has postulated a correlation of the first expression of C-gene function in teleosts with differentiation of the neural retina. Kunz (1971), using a light microscope, observed in the guppy (Lebistes retieulatus) C4 appearance at the time of photoreceptor cell differentiation of the neural retina. Nakano and Hasegawa (1971) have shown, using light microscopy, that morphological differentiation may precede the appearance of the "eye" band in the medaka by two days. Miller and Whitt (1975) observed that the retinal-specific LDH appears on the fifth day after hatching in the green sunfish, at the time when the first appearance of the C4 isozyme is tightly coupled with the morphological and probably functional differentiation of the retina. Similar results were obtained by Champion and Whitt (1976). Philipp and Whitt (1977) determined, that in the medaka the initiation of Ldh-C expression is closely coupled with morphological and functional differentiation of these cells in which this locus is predominantly expressed in the adult. Basaglia et al. (1990) determined the appearance of the "eye-specific" homotetramer LDH-C4 and of the heteropolymers B3CI, B2C2, BIC 3 at the 20th day after hatching, suggesting the concomitant function of both Ldh-B and -C genes in this time. PHYSIOLOGICALIMPLICATIONSOF LDH-C ISOZYME Detailed analyses of the physical characteristics and tissue location of the C, isozymes of several
675
Lactate dehydrogenase in teleosts
species of fishes suggest that both the structure and regulation of the teleost C gene are quite varied among different groups. Thus, many teleosts express a highly anodal isozyme restricted to the eye and brain tissues; meanwhile, gadoid and cyprinid fishes exhibit a cathodal liver-predominant C4 isozyme and a few teleost species possess C4 isozyme with characteristics somewhat intermediate between these two predominant modes. This correlation of tissuerestricted expression of an enzyme and its net charge raises a variety of questions about the distinctive role of such charge properties in neural physiology, intracellular transport, subcellular positioning and in the stabilities of such isozymes. The electrophoretic and immunochemical data (Whitt, 1987) indicate that the "eye" band (of many teleosts) and the "liver" band (of gadoids and cypriniformes) are encoded in the same genetic locus--Ldh-C. Evolutionary surveys reveal that virtually all advanced teleosts synthesize Ca isozyme in one or another tissue. Whitt and Booth (1970) localized LDH activity in the neural retina of the sword tail (Xiphophorus). Urea inactivation of the isozymes containing A and B subunits permitted the selective staining of the C4 isozyme. They found the C4 activity in the photoreceptor cells, in the region referred to as the ellipsoid. Expression of the Ldh-B locus is extremely restricted in tissues and occurs in only a few orders of teleosts, some of which are quite advanced. In the pleuronectiformes (Markert and Holmes, 1969), gasterosteiformes (Rooney and Ferguson, 1985), some zoarcids (Simonsen and Christiansen, 1985) and some umbrids (Kettler and Whitt, 1984), the Ldh-2 gene is restricted in its expression to the eye and brain instead of its typically more widespread tissue expression with predominance in the heart (Whitt, 1987). Although the precise metabolic function of different LDH isozymes has long been an unsolved problem, the physiological importance of these multiple forms has been inferred from their observed
GLyceratdehyde-5- P I (G-3-PHD I~
tissue distributions. Detailed comparison of the A4, B 4 a n d C 4 isozymes of fishes reveal that both the "eye" band and the "liver" band of the C4 tetramer differ significantly from the other two homopolymers. Therefore, the distinctive tissue distributions and biochemical properties of the C subunit suggest that this particular gene product has been specifically tailored to occupy one or more specialized metabolic niches. Because of its association with the photoreceptor cells in the retina of many teleosts, it has been hypothesized that the C4 isozyme plays a role in the retinal metabolism of teleosts (Whitt, 1970; Whitt et al., 1973c). The major biochemical process of the photoreceptor cell is the regeneration of the visual pigment. NAD ÷ serves as the cofactor for retinal dehydrogenase in the regeneration pathway in fishes (Wald, 1968). The C4 isozyme may regulate NAD+/NADH ratio in the photoreceptor cells, and thus play a key role in the vision of these teleosts. The kinetic properties of the retinal-specific LDH isozyme suggest that this isozyme might be especially suited for cells with a high constant anaerobic metabolism (Whitt et al., 1973c; Markert et al., 1975). The photoreceptor cell has been shown to have a high degree of aerobic glycolysis. The neural retina has perhaps the highest rate of oxygen utilization of any tissue. Since much of the carbohydrate metabolism of the retina is directed towards the regeneration of rhodopsin, it is proposed that the LDH-C4 isozyme may play a role in the regeneration of visual pigments in the photoreceptor cells of teleosts (Whitt, 1970). An outline of the metabolism of the visual cycle of vertebrates (modified after Wald and Hubbard, 1960) is shown in the following figure, that emphasizes the metabolic roles of LDH isozymes. NAD ÷ generated by retinal respiration and glycolysis is the coenzyme required by retinol dehydrogenase to promote rhodopsin synthesis. Thus, one of the limiting processes of rhodopsin generation is the oxidation of retinol to retinene.
Lactate RetinaL-Ca.! ~ LDH-C4~"I Is .. " " Pyruvate/S'
NADH . . . . "
I
',,
I
i NADH
cZs-Retinal NAD NADH
11 S
Pyruvate
NAD""
Mitochondria NAD
~ " ~ Respiratory
L NAD
Ic
TricarboxyLic acid cycle Fig. l.
Jr
Opsin
Rhodopsin
LMuscle-A4 # • Lactate /Heart - B ~ NADH
cis-Retina(
\/
/
676
ZULEMACOPPES
PROBABLE PARTICIPATION OF THE RETINAL-SPECIFIC LDH IN VISUAL METABOLISM
If, during certain phases of teleost metabolism, the amount of N A D + becomes limiting to the rate of rhodopsin regeneration, then an enzyme such as L D H which can convert N A D H to N A D ÷ would be important. If concentrations of pyruvate in the photoreceptor cells are relatively low, the affinity of the enzyme for substrate is of more physiological significance than its susceptibility to substrate inhibition (Whitt et al., 1973c). Thus, the high affinity for pyruvate of L D H - C 4 isozyme allows the enzyme to convert more efficiently N A D H to N A D +. The resulting N A D + would then be utilized by retinol dehydrogenase during the regeneration of rhodopsin. L D H - C 4 isozyme has a higher susceptibility to inhibition by lactate and thereby may help prevent the build-up of a deleterious concentration of this compound within the photoreceptor cells. Alternatively, the high affinity of the C4 isozyme for substrate might enable the photoreceptor cells to utilize low levels of exogenous lactate efficiently as a source of energy (Whitt et al., 1973c). Acknowledgements--The present paper was supported by the Programm of Basic Sciences (United Nations, PEDECIBA) from Uruguay. REFERENCES
Appella E. and Markert C. L. (1961) Dissociation of lactate dehydrogenase in subunits with guanidine hydrochloride. Biochem. biophys. Res. Commun. 6, 171-176. Basaglia F. (1991) Lactate dehydrogenase and their genetic variation in fifteen Sparidae species (Perciformes: Teleostei). Comp. Biochem. Physiol. 98B, 1-8. Basaglia F., Marchetti M. G. and Salvatorelli G. (1990) Genetic, developmental and comparative analysis of LDH, MDH and GPI isozymes in the sheepshead bream (Dipolodus puntazzo G.M.). Comp. Biochem Physiol. 96B, 257-266. Champion M. J. and Whitt G. S. (1976) Differential gene expression in multilocus isozyme systems of the developing green sunfish. J. exp. Zool. 196, 263-282. Coppes Z. L. (1984) Isozymes of lactate dehydrogenase in fishes of the superorder Acanthopterygii: an update. Comp. Biochem. Physiol. 79B, 14. Coppes Z., De Vecchi S,, Ferreira E. and Hirschorn M. (1990) Multilocus isozyme systems in fishes--a Minireview. Comp. Biochem. Physiol. 96B, 1-13. Coppes Z, L., Schwantes M. L. B. and Schwantes A. R. (1987) Adaptative features of enzymes from family Sciaenidae. III. Studies on lactate dehydrogenase (LDH) of fishes from the south coast of Uruguay. Comp. Biochem. Physiol. 88B, 1005 1012. Darnall D. W. and Klotz I. M. (1975) Subunit composition of proteins: a table. Archs Biochem. 166, 651-682. Fisher S. E., Shaklee J. B., Ferris S. D. and Whitt G. S. (1980) Evolution of five multilocus isozyme systems in the chordates. Genetica 52/53, 73-85. Garlick W. S. and Terwilliger R. C. (1978) The lactate dehydrogenase isozymes of six species of cottid fishes. Comp. Biochem. Physiol. 60B, 261-266. Goldberg E., Cuerrier J. P. and Ward J. C. (1969) Lactate dehydrogenase ontogeny, paternal gene activation and tetramer assembly in embryos of brook trout, lake trout and their hybrids. Biochem. Genet. 2, 335-350. Hitzeroth H., Klose J., Ohno S. and Wolf V. (1968) Asynchronous activation of parental alleles at the tissue-
specific gene loci observed in hybrid trout during early development. Biochem. Genet. 1, 287-300. Holmes R. S. (1972) Evolution of lactate dehydrogenase genes. FEBS Lett. 28, 51 55. Holt R. W. and Leibel W. S. (1987) Coexpression of distinct eye and liver specific LDH isozymes in Cichlid fish. Rapid Communication. J. exp. Zool. 244, 337-343. Kepes K. L. and Whitt G. S. (1972) Specific lactate dehydrogenase gene function in the differentiated liver of cyprinid fish. Genetics 71, 5-29. Kettler M. K. and Whitt G. S. (1984) Genetic distances among species of mudminnows. Isozyme Bull. 17, 69-71. Klebe R. J. (1975) A simple method for the quantification of isozyme patterns. Biochem. Genet. 13, 805-812. Klose J., Wolf V., Hitzeroth H., Ritter H., Atkin N. and Ohno S. (1969) Duplication of the LDH gene loci by polyploidization in the fish order Clupeiformes. Humangenetik 5, 190-196. Kunz J. (1971) Distribution of lactate dehydrogenase (and its E-isozyme) in the developing and adult retina of the guppy (Lebistes reticulatus). Rev. Suisse Zool. 78, 761-776. Lush I. E., Cowey C. B. and Knox D. (1969) The lactate dehydrogenase isozymes of twelve species of flatfish (Heterosomata). J. exp. Zool. 171, 105-118. Markert C. L. (1962) Isozymes in kidney development. In Hereditary, Developmental and Immunologic Aspects of Kidney Disease. (Edited by Metcoff I.). North-Western University Press, Evanston, IL. Markert C. L. and Faulhaber I. (1965) Lactate dehydrogenase isozyme patterns of fish. J. exp. Zool. 159, 319-332. Markert C. L. and Holmes R. S. (1969) Lactate dehydrogenase isozymes of the flatfish, Pleuronectiformes: kinetic, molecular and immunochemical analysis. J. exp. Zool. 171, 85-104. Markert C. L., Shaklee J. B. and Whitt G. S. (1975) Evolution of a gene. Science 189, 102-114. Miller E. T. and Whitt G. S. (1975) Lactate dehydrogenase isozyme synthesis and cellular differentiation in the teleost retina. In Isozymes III: Developmental Biology (Edited by Markert C. L.), pp. 359-374. Academic Press, New York. Morrison W. J. and Wright J. E. (1966) Genetic analysis of three lactate dehydrogenase isozyme systems in trout: evidence for linkage of genes coding for subunits A and B. J. exp. Zool. 163, 259-270. Nakano E. and Hasegawa M. (1971) Differentiation of the retina and retinal lactate dehydrogenase isoenzymes in the teleost Oryzias latipes. Devl. Growth Diff. 13, 351-357. Nakano E. and Whiteley A. H. (1965) Differentiation of multiple forms of four dehydrogenases in the teleost, Oryzias latipes, studied by disc electrophoresis. J. exp. Zool. 159, 167 180. Numachi K. (1972) Genetic control and subunit compositions of lactate dehydrogenase in Pseudosbora parva. Jap. J. Genet. 47, 193-201. Odense P. H. and Leung T. C. (1975) Isoelectric focusing on polyacrylamide gel and starch gel electrophoresis of some gadiform fish lactate dehydrogenase isozymes. In Isozymes III. Developmental Biology. (Edited by Markert C. L.), pp. 485-501. Academic Press, New York. Odense P. H., Leung T. C., Allen T. M. and Parker E. (1969) Multiple forms of lactate dehydrogenase in the cod, Gadus morhua L. Biochem. Genet. 3, 317-334. Ohno S. (1967) Sex-chromosomes and Sex Linked Genes. 192 pp. Springer, New York. Panepucci L. L., Schwantes M. L. B. and Schwantes A. R. (1984) Loci that encode the lactate dehydrogenase in 23 species of fish belonging to the orders Cypriniformes, Siluriformes and Perciformes: adaptative features. Comp. Biochem. Physiol. 77B, 867-876. Philipp D. P., Parker H. R. and Whitt G. S. (1983) Evolution of gene regulation: isozymic analysis of patterns of gene expression during hybrid fish development.
Lactate dehydrogenase in teleosts In Isozymes: Current Topics in Biological and Medical Research. (Edited by Ratazzi M. C., Scandalios J. G. and Whitt G. S.), Vol. 10, pp. 193-238. Alan R. Liss, New York. Philipp D. P. and Whitt G. S. (1977) Patterns of gene expression during teleost embryogenesis: lactate dehydrogenase isozyme ontogeny in the Medaka (Oryzias latipes). Devl. Biol. 59, 183-197. Rooney C. H. T. and Ferguson A. (1985) Lactate dehydrogenase isozymes and allozymes of the nine-spined stickleback Pungitus pungitus (L) (Osteichthyes: Gasterosteidae). Comp. Biochem. Physiol. 81B, 711-715. Sassaman C. and Yoshiyama R. M. (1979) Lactate dehydrogenase: A polymorphism of Anoplarchus purpurescens. J. Hered. 70, 329-334. 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: the molecular and genetic basis for the unique eye and liver LDHs of teleost fishes. J. exp. Zool. 185, 217-240. Shaklee J. B. and Whitt G. S. (1981) Lactate dehydrogenase isozymes of Gadiform fishes: divergent pattern of gene expression indicate a heterogenous taxon. Copeia 3, 563-578. Shaw C. and Barto E. (1963) Genetic evidence for the subunit structure of lactate dehydrogenase isozymes. Proc. natn. Acad. Sci. U.S.A. 50, 211-214. Simonsen V. and Christiansen F. B. (1985) Genetic of Zoarces populations. XIV. Variation of the lactate dehydrogenase isoenzymes. Hereditas 103, 177-185. Smith P. J. and Jamieson A. (1978) Enzyme polymorphism in the Atlantic mackerel, Scomber scomber L. Comp. Biochem. Physiol. 60B, 487-489. Utter F. M. and Hodgins H. O. (1969) Lactate dehydrogenase isozymes of Pacific hake (Merluccius productus). J. exp. Zool. 172, 59-67. Wald G. (1968) Molecular basis of visual excitation. Science 162, 230-239. Wald G. and Hubbard M. (1960) Enzymes aspects of the visual processes. In The Enzymes (Edited by Boyer et al.), Vol. 3, pp. 369-386. Academic Press, New York. Whitt G. S. (1968) Developmental genetics of lactate dehydrogenase isozymes unique to eye and brain in teleosts. Genetics 60, 237 (Abstract). Whitt G. S. (1969) Homology of lactate dehydrogenase genes. E function in the teleost nervous system. Science 166, 1156-1158. Whitt G. S. (1970) Developmental genetics of the lactate dehydrogenase isozymes of fish. J. exp. Zool. 175, 1-36.
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Whitt G. S. (1975) A unique lactate dehydrogenase isozyme in the teleost retina. In Vision in Fishes. (Edited by Ali M. A.), pp. 459-470. Plenum Press, New York. Whitt G. S. (1981a) Evolution of isozyme loci and their differential regulation In Evolution Today, Proceedings of the Second International Congress of Systematics and Evolutionary Biology. (Edited by Scudder G. G. E. and Reveal J. L.), pp. 271-289. Hunt Institute for Botanical Documentation. Whitt G. S. (1981b) Developmental genetics of fishes: isozyme analysis of differential gene expression. Am. Zool. 21, 549-572. Whitt G. S. (1987) Species differences in isozyme tissue patterns: their utility for systematic and evolutionary analysis. In Isozymes: Current Topics and Medical Research. Genetics, Development and Evolution. Vol. 15, pp. 1-26. Alan R. Liss, New York. Whitt G. S. and Booth G. M. (1970) Localization of lactate dehydrogenase in the cells of the fish (Xiphophorus helleri) eye. J. exp. Zool. 174, 215-224. Whitt G. S., Childers W. F. and Wheat T. E. (1971) The inheritance of tissue-specific LDH isozymes in interspecific bass (Micropterus) Hybrids. Biochem. Genet. 5, 257-273. Whitt G. S., Childers W. F. and Cho P. L. (1973a) Allelic expression at enzyme loci in an interbridal hybrid sunfish. J. Hered. 64, 55-61. Whitt G. S., Childers W. F., Tranquilli J. and Champion M. (1973b) Extensive heterozygosity at three loci in hybrid sunfish populations. Biochem. Genet. 8, 55-72. Whitt G. S., Miller E. T. and Shaklee J. B. (1973c) Developmental and biochemical genetics of lactate dehydrogenase insozyme in fishes. In Genetics and Mutagenesis of Fish. (Edited by Schroeder J. M.), pp. 243-276. Springer, Berlin. Whitt G. S., Shaklec J. B. and Markert C. L. (1975) Evolution of the lactate dehydrogenase isozymes of fishes. In Isozymes IV: Genetics and Evolution. (Edited by Markert C. L.), pp. 381-400. Academic Press, New York. Wilson A. C., Kaplan N. O., Levine L., Pesce A., Reichlin M. and Allison W. S. (1964) Evolution of lactive dehydrogenase. FASEB Fedn Proc. 23, 1258-1266. Wilson F. R., Whitt G. S. and Ladd Prosser C. (1973) Lactate dehydrogenase and malate dehydrogenase isozyme patterns in tissues of temperature-acclimated goldfish (Carassius auratus L). Comp. Biochem. Physiol. 46B, 105-116. Wiseman D. E., Echelle A. and Echelle A. F. (1978) Electrophoretic evidence for subspecific differentiation and intergradiation in Etheostoma spectabile (Teleostei, Percidae). Copeia 2, 320-327.
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
Printed in Great Britain
MINI REVIEW LACTATE DEHYDROGENASE IN TELEOSTS. THE ROLE OF LDH-C4 ISOZYME ZULEMA COPPES
Cfitedra de Bioquimica, Facultad de Quimica, General Flores 2124, Montevideo, Uruguay (Fax: 00598-2-941-906) (Received 23 September 1991)
Abstract--1. Lactate dehydrogenase (LDH) occupies an important position in cell metabolism. 2. Teleosts possess at least three genetic loci coding for lactate dehydrogenase subunits, Ldh-A, Ldh-B and Ldh-c. LDH exists in most tissues in several isozymic forms. 3. The isozyme LDH-C4 is synthesized predominantly in regions of the nervous system concerned with the eye.
INTRODUCTION
Several examples of homologous enzymes have been described in fishes: creatine kinase (CK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI) (Coppes et al., 1990). These multilocus isozyme systems were formed by relatively ancient gene duplications and are suitable candidates for the study of evolution of isozymic loci (Whitt, 1981 a). The genetic and molecular bases that give rise to their isozyme distribution are very well known. The number of isozymic loci present in a species is easily estimated by electrophoresis. Such loci diverged in most cases hundreds of millions of years ago. Thus, their isozymes differ considerably owing to innumerable amino acid substitutions. A corroboration of the number of loci present is obtained by the presence of genetic variants, and by the observation of the number and relative intensities of isozymes, as well as their physical and antigenic properties (Fisher et al., 1980; Whitt, 1981a). Lactate dehydrogenase is an isozyme system which, owing to its multigenic nature, is an interesting model for evolutionary studies, and from this aspect has been widely investigated (Wilson et al., 1964; Holmes, 1972; Whitt et al., 1973a,b, 1975; Markert et al., 1975; Fisher et al., 1980; Whitt, 1981b, 1987; Coppes, 1984; Coppes et al., 1990). In advanced fishes LDH is codified by three loci, Ldh-A, Ldh-B and Ldh-C. Ldh-C is restricted in its expression to neural tissues, eye and brain (in most advanced fishes) or liver (in some fishes). The present paper discusses the localization of the LDH-C4 isozyme in tissues of advanced fishes and tries to infer its function in retinal metabolism. LACTATE DEHYDROGENASE ISOZYMES
IN FISHES
Lactate dehydrogenase (lactate: N A D oxidoreductase, EC 1.1.1.27) with a tetrameric structure and a mol. wt of 14,000 (Darnall and Klotz, 1975)
catalyzes the interconversion of lactate and pyruvate. The reaction serves as an important source of oxidized coenzyme (nicotinamide adenine dinucleotide NAD +) during periods of transient anaerobiosis, or of a reduced form of such a coenzyme during aerobiosis. The existence of multiple loci encoding LDH has been well-documented for almost all vertebrate types (Coppes et al., 1990). In invertebrates, the enzymes catalyzing the pyruvate-lactate interconversion are not yet well characterized. In vertebrates, LDH is known to comprise a single homologous family (Markert et al., 1975). This family exists in a variety of isozymic forms, all related in that they catalyze the same chemical reaction, but all different from one another in molecular structure and commonly in genetic control. In vertebrates the polypeptide subunits of LDH are encoded in at least two genetic loci whose alleles are codominantly expressed (Markert, 1962; Shaw and Barto, 1963). According to Markert et al. (1975) only a single gene encoded the LDH polypeptide at the beginning of vertebrate evolution. The subunits encoded by this gene are able to polymerize to make a homotetramer with properties similar to those of the A4 isozyme commonly occurring in the skeletal muscle of all vertebrates. All contemporary vertebrates examined, save one, the Lampreia, have at least two genes coding for LDH polypeptides. The original A gene must have soon duplicated (Markert et al., 1975); later, these two A-like genes diverged by mutations to give rise to two distinctly different genes now designated A and B (Ohno, 1967; Markert et al., 1975; Whitt et al., 1975), which associate at random to yield binomial distributions of the five tetrameric isozymes (Appella and Markert, 1961; Markert et al., 1975). A third LDH locus has been found in many Orders of fish; the product of this third locus is referred to as the C4 isozyme (Shaklee et aL, 1973). This C 4 isozyme is like the B 4 isozyme in possessing a lower substrate concentration optimum and greater susceptibility to substrate inhibition than A4 isozyme in vitro 673
674
ZULEMA COPPES
(Shaklee et al., 1973). It is probable that these kinetic properties make the B 4 and C4 isozymes suited to tissues which are relatively more aerobic (Markert and Whitt, 1975). LDH-C ISOZYME IN FISHES
The Ldh-C locus present in most Actinopterygii (Coppes et al., 1990) was first observed in Chondrosteans (Fisher et aL, 1980). The Ldh-C locus probably arose from duplication of an Ldh-B locus (Whitt, 1969, 1970). In the primitive teleosts possessing a Ldh-C locus (Acipenseriformes, Amiiformes, Elopiformes, Anguilliformes and Osteoglossiformes) LDH-C4 isozyme is present in a variety of tissues (Markert et al., 1975; Whitt et al., 1975). LDH-C expression tends to be parallel to LDH-B in some primitive groups of teleosts (Fisher et al., 1980). In the advanced teleosts the structure and function of Ldh-C locus evolved in two different ways. In most Orders the gene encodes an anodic isozyme which predominates in "eye" and brain (Whitt, 1970, 1981a,b; Whitt et al., 1971, 1973b; Markert et al., 1975; Philipp and Whitt, 1977; Wiseman et al., 1978; Sassaman and Yoshiyama, 1979; Smith and Jamieson, 1978; Philipp et al., 1983; Paneppucci et al., 1984; Coppes et al., 1987; Basaglia, 1991). In some species of Cypriniformes and Gadiformes, the Ldh-C locus encodes a cathodic isozyme which predominates in liver (Markert and Faulhaber, 1965; Klose et al., 1969; Lush et al., 1969; Odense et al., 1969; Utter and Hodgins, 1969; Kepes and Whitt, 1972; Numachi, 1972; Sensabaugh and Kaplan, 1972; Whitt et al., 1973c; Wilson et al., 1973; Odense and Leung, 1975; Shaklee and Whitt, 1981; Coppes et al., 1990). The discovery of several fish species in which biochemically distinct eye- and liver-specific LDH isozymes are coexpressed, is discussed in the light of the currently accepted hypothesis that these two isozymes are encoded by a single locus (Ldh-C) which has undergone divergent tissue expression in several other major groups of teleosts (Holt and Leibel, 1987). The association between B and C subunits detected in neural tissues, commonly found in the literature, show that when a heteropolimer is present, it consists of B and C subunits (Morrison and Wright, 1966; Odense et al., 1969; Whitt, 1968, 1969, 1970, 1981b; Kepes and Whitt, 1972; Shaklee et al., 1973; Philipp and Whitt, 1977; Garlick and Terwilliger, 1978; Shaklee and Whitt, 1981; Coppes et al., 1987; Basaglia et al., 1990). Taking into account the physical, kinetic and immunochemical properties the C subunit in fishes, as well as in mammals and birds, is more similar to the B than to the A subunit. However, according to Whitt (1987), the data are not sufficient to decide whether Ldh-C gene is more closely related to A or to B. Shaklee et al. (1973) and Garlick and Terwilliger (1978) describe some species of fishes where associations of A and C subunits are detected. The results obtained by Coppes et al. (1987) suggest [through the method of serial dilutions of Klebe (1975)] that the C subunit of neural tissues is more similar to the B than to the A subunit.
DEVELOPMENTAL STUDIES
Tissue-specific isozyme patterns in the adult organism reflect developmental changes that occur in the control of gene expression. The presence of three homologous LDH loci in the teleost provides a system for examining this differential expression. The L D H - C 4 isozyme of teleosts is one example of an isozyme which is often restricted to a single cell type. In most species of teleosts, the Ldh-C gene appears to be expressed in the photoreceptor cells of the neural retina (Miller and Whitt, 1975). Thus, it has been possible to determine that the expression of the Ldh-C gene is tightly coordinated with the morphological and functional differentiation of the neural retina (Whitt, 1975). So, if LDH-C4 is important to retinal metabolism, its appearance during embryogenesis should occur concomitantly with morphological and functional differentiation of the retina. Thus, the timing of the synthesis of this retinal-specific isozyme with respect to the timing of the morphological differentiation was determined. Nakano and Whiteley (1965) reported that the "eye" band appears abruptly at hatching in the medaka (Oryzias latipes): they concluded that this was due to the conversion from anaerobic to an aerobic metabolism at hatching. Whitt (1970) observed the "eye" band at least 24 hr before the time of hatching in another teleost, Fundulus heteroelitus, and suggested that hatching was not the necessary stimulus to C-gene activation in this fish. In the trout, Hitzeroth et al. (1968) and Goldberg et al. (1969) showed that the retinal-specific LDH did not appear until a considerable time after hatching. Whitt (1968, 1970) has postulated a correlation of the first expression of C-gene function in teleosts with differentiation of the neural retina. Kunz (1971), using a light microscope, observed in the guppy (Lebistes retieulatus) C4 appearance at the time of photoreceptor cell differentiation of the neural retina. Nakano and Hasegawa (1971) have shown, using light microscopy, that morphological differentiation may precede the appearance of the "eye" band in the medaka by two days. Miller and Whitt (1975) observed that the retinal-specific LDH appears on the fifth day after hatching in the green sunfish, at the time when the first appearance of the C4 isozyme is tightly coupled with the morphological and probably functional differentiation of the retina. Similar results were obtained by Champion and Whitt (1976). Philipp and Whitt (1977) determined, that in the medaka the initiation of Ldh-C expression is closely coupled with morphological and functional differentiation of these cells in which this locus is predominantly expressed in the adult. Basaglia et al. (1990) determined the appearance of the "eye-specific" homotetramer LDH-C4 and of the heteropolymers B3CI, B2C2, BIC 3 at the 20th day after hatching, suggesting the concomitant function of both Ldh-B and -C genes in this time. PHYSIOLOGICALIMPLICATIONSOF LDH-C ISOZYME Detailed analyses of the physical characteristics and tissue location of the C, isozymes of several
675
Lactate dehydrogenase in teleosts
species of fishes suggest that both the structure and regulation of the teleost C gene are quite varied among different groups. Thus, many teleosts express a highly anodal isozyme restricted to the eye and brain tissues; meanwhile, gadoid and cyprinid fishes exhibit a cathodal liver-predominant C4 isozyme and a few teleost species possess C4 isozyme with characteristics somewhat intermediate between these two predominant modes. This correlation of tissuerestricted expression of an enzyme and its net charge raises a variety of questions about the distinctive role of such charge properties in neural physiology, intracellular transport, subcellular positioning and in the stabilities of such isozymes. The electrophoretic and immunochemical data (Whitt, 1987) indicate that the "eye" band (of many teleosts) and the "liver" band (of gadoids and cypriniformes) are encoded in the same genetic locus--Ldh-C. Evolutionary surveys reveal that virtually all advanced teleosts synthesize Ca isozyme in one or another tissue. Whitt and Booth (1970) localized LDH activity in the neural retina of the sword tail (Xiphophorus). Urea inactivation of the isozymes containing A and B subunits permitted the selective staining of the C4 isozyme. They found the C4 activity in the photoreceptor cells, in the region referred to as the ellipsoid. Expression of the Ldh-B locus is extremely restricted in tissues and occurs in only a few orders of teleosts, some of which are quite advanced. In the pleuronectiformes (Markert and Holmes, 1969), gasterosteiformes (Rooney and Ferguson, 1985), some zoarcids (Simonsen and Christiansen, 1985) and some umbrids (Kettler and Whitt, 1984), the Ldh-2 gene is restricted in its expression to the eye and brain instead of its typically more widespread tissue expression with predominance in the heart (Whitt, 1987). Although the precise metabolic function of different LDH isozymes has long been an unsolved problem, the physiological importance of these multiple forms has been inferred from their observed
GLyceratdehyde-5- P I (G-3-PHD I~
tissue distributions. Detailed comparison of the A4, B 4 a n d C 4 isozymes of fishes reveal that both the "eye" band and the "liver" band of the C4 tetramer differ significantly from the other two homopolymers. Therefore, the distinctive tissue distributions and biochemical properties of the C subunit suggest that this particular gene product has been specifically tailored to occupy one or more specialized metabolic niches. Because of its association with the photoreceptor cells in the retina of many teleosts, it has been hypothesized that the C4 isozyme plays a role in the retinal metabolism of teleosts (Whitt, 1970; Whitt et al., 1973c). The major biochemical process of the photoreceptor cell is the regeneration of the visual pigment. NAD ÷ serves as the cofactor for retinal dehydrogenase in the regeneration pathway in fishes (Wald, 1968). The C4 isozyme may regulate NAD+/NADH ratio in the photoreceptor cells, and thus play a key role in the vision of these teleosts. The kinetic properties of the retinal-specific LDH isozyme suggest that this isozyme might be especially suited for cells with a high constant anaerobic metabolism (Whitt et al., 1973c; Markert et al., 1975). The photoreceptor cell has been shown to have a high degree of aerobic glycolysis. The neural retina has perhaps the highest rate of oxygen utilization of any tissue. Since much of the carbohydrate metabolism of the retina is directed towards the regeneration of rhodopsin, it is proposed that the LDH-C4 isozyme may play a role in the regeneration of visual pigments in the photoreceptor cells of teleosts (Whitt, 1970). An outline of the metabolism of the visual cycle of vertebrates (modified after Wald and Hubbard, 1960) is shown in the following figure, that emphasizes the metabolic roles of LDH isozymes. NAD ÷ generated by retinal respiration and glycolysis is the coenzyme required by retinol dehydrogenase to promote rhodopsin synthesis. Thus, one of the limiting processes of rhodopsin generation is the oxidation of retinol to retinene.
Lactate RetinaL-Ca.! ~ LDH-C4~"I Is .. " " Pyruvate/S'
NADH . . . . "
I
',,
I
i NADH
cZs-Retinal NAD NADH
11 S
Pyruvate
NAD""
Mitochondria NAD
~ " ~ Respiratory
L NAD
Ic
TricarboxyLic acid cycle Fig. l.
Jr
Opsin
Rhodopsin
LMuscle-A4 # • Lactate /Heart - B ~ NADH
cis-Retina(
\/
/
676
ZULEMACOPPES
PROBABLE PARTICIPATION OF THE RETINAL-SPECIFIC LDH IN VISUAL METABOLISM
If, during certain phases of teleost metabolism, the amount of N A D + becomes limiting to the rate of rhodopsin regeneration, then an enzyme such as L D H which can convert N A D H to N A D ÷ would be important. If concentrations of pyruvate in the photoreceptor cells are relatively low, the affinity of the enzyme for substrate is of more physiological significance than its susceptibility to substrate inhibition (Whitt et al., 1973c). Thus, the high affinity for pyruvate of L D H - C 4 isozyme allows the enzyme to convert more efficiently N A D H to N A D +. The resulting N A D + would then be utilized by retinol dehydrogenase during the regeneration of rhodopsin. L D H - C 4 isozyme has a higher susceptibility to inhibition by lactate and thereby may help prevent the build-up of a deleterious concentration of this compound within the photoreceptor cells. Alternatively, the high affinity of the C4 isozyme for substrate might enable the photoreceptor cells to utilize low levels of exogenous lactate efficiently as a source of energy (Whitt et al., 1973c). Acknowledgements--The present paper was supported by the Programm of Basic Sciences (United Nations, PEDECIBA) from Uruguay. REFERENCES
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specific gene loci observed in hybrid trout during early development. Biochem. Genet. 1, 287-300. Holmes R. S. (1972) Evolution of lactate dehydrogenase genes. FEBS Lett. 28, 51 55. Holt R. W. and Leibel W. S. (1987) Coexpression of distinct eye and liver specific LDH isozymes in Cichlid fish. Rapid Communication. J. exp. Zool. 244, 337-343. Kepes K. L. and Whitt G. S. (1972) Specific lactate dehydrogenase gene function in the differentiated liver of cyprinid fish. Genetics 71, 5-29. Kettler M. K. and Whitt G. S. (1984) Genetic distances among species of mudminnows. Isozyme Bull. 17, 69-71. Klebe R. J. (1975) A simple method for the quantification of isozyme patterns. Biochem. Genet. 13, 805-812. Klose J., Wolf V., Hitzeroth H., Ritter H., Atkin N. and Ohno S. (1969) Duplication of the LDH gene loci by polyploidization in the fish order Clupeiformes. Humangenetik 5, 190-196. Kunz J. (1971) Distribution of lactate dehydrogenase (and its E-isozyme) in the developing and adult retina of the guppy (Lebistes reticulatus). Rev. Suisse Zool. 78, 761-776. Lush I. E., Cowey C. B. and Knox D. (1969) The lactate dehydrogenase isozymes of twelve species of flatfish (Heterosomata). J. exp. Zool. 171, 105-118. Markert C. L. (1962) Isozymes in kidney development. In Hereditary, Developmental and Immunologic Aspects of Kidney Disease. (Edited by Metcoff I.). North-Western University Press, Evanston, IL. Markert C. L. and Faulhaber I. (1965) Lactate dehydrogenase isozyme patterns of fish. J. exp. Zool. 159, 319-332. Markert C. L. and Holmes R. S. (1969) Lactate dehydrogenase isozymes of the flatfish, Pleuronectiformes: kinetic, molecular and immunochemical analysis. J. exp. Zool. 171, 85-104. Markert C. L., Shaklee J. B. and Whitt G. S. (1975) Evolution of a gene. Science 189, 102-114. Miller E. T. and Whitt G. S. (1975) Lactate dehydrogenase isozyme synthesis and cellular differentiation in the teleost retina. In Isozymes III: Developmental Biology (Edited by Markert C. L.), pp. 359-374. Academic Press, New York. Morrison W. J. and Wright J. E. (1966) Genetic analysis of three lactate dehydrogenase isozyme systems in trout: evidence for linkage of genes coding for subunits A and B. J. exp. Zool. 163, 259-270. Nakano E. and Hasegawa M. (1971) Differentiation of the retina and retinal lactate dehydrogenase isoenzymes in the teleost Oryzias latipes. Devl. Growth Diff. 13, 351-357. Nakano E. and Whiteley A. H. (1965) Differentiation of multiple forms of four dehydrogenases in the teleost, Oryzias latipes, studied by disc electrophoresis. J. exp. Zool. 159, 167 180. Numachi K. (1972) Genetic control and subunit compositions of lactate dehydrogenase in Pseudosbora parva. Jap. J. Genet. 47, 193-201. Odense P. H. and Leung T. C. (1975) Isoelectric focusing on polyacrylamide gel and starch gel electrophoresis of some gadiform fish lactate dehydrogenase isozymes. In Isozymes III. Developmental Biology. (Edited by Markert C. L.), pp. 485-501. Academic Press, New York. Odense P. H., Leung T. C., Allen T. M. and Parker E. (1969) Multiple forms of lactate dehydrogenase in the cod, Gadus morhua L. Biochem. Genet. 3, 317-334. Ohno S. (1967) Sex-chromosomes and Sex Linked Genes. 192 pp. Springer, New York. Panepucci L. L., Schwantes M. L. B. and Schwantes A. R. (1984) Loci that encode the lactate dehydrogenase in 23 species of fish belonging to the orders Cypriniformes, Siluriformes and Perciformes: adaptative features. Comp. Biochem. Physiol. 77B, 867-876. Philipp D. P., Parker H. R. and Whitt G. S. (1983) Evolution of gene regulation: isozymic analysis of patterns of gene expression during hybrid fish development.
Lactate dehydrogenase in teleosts In Isozymes: Current Topics in Biological and Medical Research. (Edited by Ratazzi M. C., Scandalios J. G. and Whitt G. S.), Vol. 10, pp. 193-238. Alan R. Liss, New York. Philipp D. P. and Whitt G. S. (1977) Patterns of gene expression during teleost embryogenesis: lactate dehydrogenase isozyme ontogeny in the Medaka (Oryzias latipes). Devl. Biol. 59, 183-197. Rooney C. H. T. and Ferguson A. (1985) Lactate dehydrogenase isozymes and allozymes of the nine-spined stickleback Pungitus pungitus (L) (Osteichthyes: Gasterosteidae). Comp. Biochem. Physiol. 81B, 711-715. Sassaman C. and Yoshiyama R. M. (1979) Lactate dehydrogenase: A polymorphism of Anoplarchus purpurescens. J. Hered. 70, 329-334. 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: the molecular and genetic basis for the unique eye and liver LDHs of teleost fishes. J. exp. Zool. 185, 217-240. Shaklee J. B. and Whitt G. S. (1981) Lactate dehydrogenase isozymes of Gadiform fishes: divergent pattern of gene expression indicate a heterogenous taxon. Copeia 3, 563-578. Shaw C. and Barto E. (1963) Genetic evidence for the subunit structure of lactate dehydrogenase isozymes. Proc. natn. Acad. Sci. U.S.A. 50, 211-214. Simonsen V. and Christiansen F. B. (1985) Genetic of Zoarces populations. XIV. Variation of the lactate dehydrogenase isoenzymes. Hereditas 103, 177-185. Smith P. J. and Jamieson A. (1978) Enzyme polymorphism in the Atlantic mackerel, Scomber scomber L. Comp. Biochem. Physiol. 60B, 487-489. Utter F. M. and Hodgins H. O. (1969) Lactate dehydrogenase isozymes of Pacific hake (Merluccius productus). J. exp. Zool. 172, 59-67. Wald G. (1968) Molecular basis of visual excitation. Science 162, 230-239. Wald G. and Hubbard M. (1960) Enzymes aspects of the visual processes. In The Enzymes (Edited by Boyer et al.), Vol. 3, pp. 369-386. Academic Press, New York. Whitt G. S. (1968) Developmental genetics of lactate dehydrogenase isozymes unique to eye and brain in teleosts. Genetics 60, 237 (Abstract). Whitt G. S. (1969) Homology of lactate dehydrogenase genes. E function in the teleost nervous system. Science 166, 1156-1158. Whitt G. S. (1970) Developmental genetics of the lactate dehydrogenase isozymes of fish. J. exp. Zool. 175, 1-36.
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