Adaptative features of enzymes from family sciaenidae (Perciformes)--I. Studies on soluble malate dehydrogenase (s-MDH) and creatinine kinase (CK) of fishes from the south coast of Uruguay

1. Electrophoretic analysis of the soluble malate dehydrogenase (s-MDH) and creatine kinase (CK) isozyme patterns of three species of temperate fishes (Scianidae, Perciformes) indicates at least two loci for s-MDH, Mdh-A and Mdh-B, and four CK, Ck-A, Ck-B, Ck-C and Ck-D. 2. The subunits encoded by these loci occurred at different levels in different tissues and organs analyzed. 3. Through electrophoretic analysis the products of these loci showed different behaviour to changes in temperature. 4. Relative activities of s-MDH and CK isozymes were compared by Klebe's (1975) method to determine pattern of divergence of duplicated gene expression in the three studied species.     

Isozymes of glucosephosphate isomerase (PGI) in fishes of the subclass actinopterygii

A compilation of the species of fishes of the subclass Actinopterygii for the study of the PGI isozyme system is given. PGI appears to be codified by more than one locus in fishes; 65% of the species analysed here have two loci for PGI. PGI duplication in fishes and the relationship of isozymes of PGI with temperature and metabolism are discussed.     

An immunochemical analysis of the novel liver-restricted LDH activity from cichlid fishes (Cichlidae: Teleostei) confirms its non-orthology with piscine LDH-C

In an electrophoretic survey of lactate dehydrogenase (LDH) isozymes in neotropical cichlid fishes (Perciformes: Cichlidae) several species were discovered in which a cathodal liver-restricted isozyme is expressed along with the highly anodal eye-restricted isozyme (LDH-C₄) typically encountered in perciform fishes. Biochemical characterization of these two isozymes from the basketmouth cichlid, Acaronia nassa (Heckel), strongly suggested that they were non-orthologous and challenged the accepted view that eye- and liver-restricted LDH isozymes are alternative expressions of the same (LDH-C *) gene. In this study, antiserum raised against cypriniform (goldfish) liver-restricted LDH-C₄ failed to cross-react with the basketmouth liver-restricted analogue while effectively titrating the eye-restricted, anodal isozyme and, at higher titres, the LDH-B₄, heart-restricted isozyme from all cichlid species. Anti-serum raised against basketmouth muscle-restricted LDH-A₄ failed to titrate any of the eye- and liver-restricted isozymes. These data confirm the orthology of eye- and liver-restricted LDH isozymes in Cypriniform and Perciform fishes as originally proposed, suggest that the liver-restricted isozyme of cichlid fishes is non-orthologous and further raise the question of identity and evolutionary origin of this anomalous LDH activity.     

Subunit hybridization and immunological studies of duplicated phosphoglucose isomerase isozymes

We examined structural and functional properties of the recently duplicated cytosolic isozymes of phosphoglucose isomerase (PGI) (EC 5.3.1.9) of the wild plant Clarkia xantiana and related species. A new purification protocol yielded PGIs with high specific activities. The duplicated PGI isozymes showed similar substrate affinities (apparent Km) in both catalytic directions. A newly devised competitive enzyme-linked immunosorbent assay (ELISA), using polyclonal antibodies, distinguished sodium dodecyl sulfate (SDS)-denatured PGIs coded by the duplicated loci but did not discriminate between allelic products of one of the loci. In vitro dissociation and reassociation experiments revealed that the duplicated subunits differed in their efficiency of reassociation. The difference was closely correlated with differences between the isozymes in their in vivo accumulation. In contrast, allelic subunits in species with or without the duplication were able to reassociate with similar efficiency. The duplicated PGI isozymes have diverged more in structural properties than kinetic ones. The total evidence suggests that mechanisms have evolved which reduce the potential consequences of the duplication.     

Adaptative features of enzymes from family Sciaenidae--III. Studies on lactate dehydrogenase (LDH) of fishes from the south coast of Uruguay

1. Electrophoretic analyses of lactate dehydrogenase isozyme patterns of three species of temperate fish belonging to family Sciaenidae (order Perciformes) indicates that at least three LDH loci--Ldh-A, Ldh-B and Ldh-C are active. 2. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 3. The products of these loci show different response to changes in temperature. The results were obtained by electrophoretic analyses. 4. Thermostability of skeletal muscle, heart and eye lactate dehydrogenase incubated at 60 degrees C for different periods of time were examined. 5. Relative activities of LDH isozymes were compared by Klebe's method to determine pattern of divergence of duplicated gene expression in the three species studied.     

Coexpression of distinct eye- and liver-specific LDH isozymes in cichlid fish

In a recent electrophoretic survey of lactate dehydrogenase (LDH) in neotropical cichlid fishes (Perciformes, Cichlidae) we have discovered several species in which a cathodal liver-specific isozyme is expressed along with the highly-anodal eye-specific isozyme (LDH-C4) typically encountered in perciform fishes. We believe this fourth, liver-specific LDH isozyme to be real and not artifactual since homogenization of fresh liver from one of these species, the Basketmouth cichlid (Acaronia nassa), in either of two nondenaturing detergents or in the presence of the protease inhibitor phenylmethylsulfonylfluoride affects neither the presence nor mobility of this cathodal band. Moreover, it continues to be expressed in the captively bred F1 of these same wild fish. The discovery of several fish species, like the Basketmouth, in which biochemically distinct eye- and liver-specific LDH isozymes are coexpressed, is discussed in 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 teleost groups. Preliminary characterization of the liver-specific isozyme relative to the eye-specific LDH-C4 in the Basketmouth cichlid with respect to thermolability and NADH-induced binding to oxamate-sepharose columns suggests that the eye- and liver-specific LDH isozymes are biochemically quite distinct in this fish and that they are probably encoded by two distinct loci.     

Some aspects of isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in fish

1. The present paper reports some aspects of the isozymes of LDH, MDH and GPI in fish. 2. In Petromyzontiformes LDH is encoded by a single Ldh-A gene locus. In Myxiniformes and in most vertebrates LDH is encoded by two gene loci, A and B. A third Ldh-C locus is characteristic of the bony fishes Actinopterygii. 3. In fish the MDH isozymes are generally encoded by three gene loci Mdh-M, Mdh-A and Mdh-B. 4. In most diploid bony fish the GPI is controlled by two independent gene loci Gpi-A and Gpi-B. 5. The relationships of isozymes with evolution of vertebrates, tissual specificity, ontogenetic changes, with physiological and metabolic roles are discussed.     

Phosphoglucose isomerase gene duplication in the bony fishes: An evolutionary history

Electrophoretic patterns of phosphoglucose isomerase (PGI) in bony fishes provide strong evidence for a model of genetic control by two independent structural gene loci, most likely resulting from a gene duplication. This model is confirmed by a comparison of certain kinetic and molecular properties of the PGI homodimers (PGI-1 and PGI-2) isolated from extracts of the teleost Astyanax mexicanus. In addition, in most higher teleosts examined, the PGI enzymes show a regular pattern of tissue distribution, with PGI-2 predominant in muscle, the heterodimer often strongest in the heart, and PGI-1 predominant in liver and other organs. An examination of 53 species of bony fishes belonging to 38 families indicates a widespread occurrence of duplicate PGI loci and an early origin of the gene duplication, perhaps in the Leptolepiformes. The apparent presence of three PGI loci in trout and goldfish exemplifies how new loci can be incorporated into the genome through polyploidization.This research was supported in part by a NSF graduate traineeship to J.C.A., by the Clayton Foundation for Research in Biochemistry (G.B.K.), by NSF Grant GB-15644 and NIH Grant GM-15769 to Robert K. Selander, and by contract AT(38-1)-310 between the University of Georgia and the U.S. Atomic Energy Commission.     

Peroxidase and leucine-aminopeptidase in diploidMedicago species closely related to alfalfa: Multiple gene loci,multiple allelism,and linkage

Summary The genetics of peroxidase and leucine-aminopeptidase isozymes was studied utilizing starch gel electrophoresis in the diploidsMedicago sativa L. (M. coerulea Less.) andM. falcata L. Three anodal and one cathodal sets of peroxidase isozymes identify four linked loci. In addition, two anodal sets of leucine-aminopeptidase isozymes identify two loci that may be linked. The allozymes at each of the loci segregated as expected for monomeric enzymes. However in several crosses there were deficiencies in the number of progeny in particular genotypic classes. This could result from the segregation of recessive deleterious genes linked to some of the allozyme alleles. This is the first report of multiple loci and multiple alleles determining isozymes inMedicago. Supported by grants from the Alberta Research Council (No. D1B02 to R.C. von Borstel) and the Computer Use and Policy Committee, University of Alberta     

PHYLOGENETIC IMPLICATIONS OF DIFFERENCES IN NUMBER OF PLASTID PHOSPHOGLUCOSE ISOMERASE ISOZYMES IN NORTH AMERICAN COREOPSIS (ASTERACEAE: HELIANTHEAE: COREOPSIDINAE)

Enzyme electrophoresis was employed to ascertain the number of loci encoding plastid phosphoglucose isomerase (PGI) in species representing all sections of North American Coreopsis. Several species from each of the closely related genera Bidens, Coreocarpus, Cosmos, and Thelesperma were also examined. Species in nine of the 11 sections of North American Coreopsis have two isozymes for plastid PGI, and nearly all species examined in the four other genera also have two (one species has three) isozymes. Since most diploid vascular plants have one plastid PGI isozyme, a gene duplication probably occurred in an ancestor that is common to Coreopsis and the other four genera. That is, two isozymes represent the ancestral number for Coreopsis. The two sections (Electra and Anathysana) apparently lacking the duplication are closely related woody plants restricted largely to Mexico. One gene encoding plastid PGI ostensibly was silenced in a common ancestor of these two sections. This is concordant with other data suggesting a close relationship between the two sections, i.e., they appear to represent a monophyletic group. The electrophoretic data also indicate that 1) the enigmatic monotypic section Silphidium is more closely related to eastern North American sections and not derived from section Electra; and 2) section Anathysana is not ancestral to the three California sections Leptosyne, Pugiopappus, and Tuckermannia; rather, it represents a terminal element closely related to and possibly derived from section Electra.     

Temperature-dependent kinetic variation among phosphoglucose isomerase allozymes from the wing-polymorphic water strider, Limnoporus canaliculatus

Phosphoglucose isomerase (PGI) allozymes were isolated from the wing- polymorphic water strider, Limnoporus canaliculatus, and were characterized biochemically with respect to temperature-dependent kinetic and thermostability properties. At higher temperatures, the allozymes exhibited significant differences in Michaelis constant (Km) values for substrates of both the forward and reverse reaction directions. Results were consistent with expectations of adaptive kinetic differentiation based on the latitudinal variation of PGI allele frequencies. PGI genotypes also differed with regard to maximal velocity (Vmax)/Km ratios at higher temperatures. These differences were due primarily, if not exclusively, to allozyme-dependent variation in Km values. The allozymes also exhibited dramatic differences in thermostability. However, no thermostability differences were observed when the substrate analogue 6-phosphogluconate was present in the incubation medium. The data from this study, together with data from Mytilus edulis and Metridium senile on temperature-dependent kinetic variation among PGI allozymes, form a consistent picture of natural selection influencing the clinal variation of alleles at this locus in these three phylogenetically distant organisms. More definitive support of this hypothesis, however, must await additional studies on the physiological effects of the allozymic variation as well as direct measurements of fitness differences among the enzyme genotypes.      

Associations of genes encoding allozymes peroxidase and superoxide dismutase in poplar and spruce species

Summary Isozymes of peroxidase (PER) and superoxide dismutase (SOD) were analyzed in vegetative buds or very young leaves of seven species and two interspecific hybrids of Populus, in progenies of seven controlled crosses of three Populus species, and in needles of five Picea species and one putative hybrid. One to three PER, and one or two SOD zones of activity were observed. Electrophoretic mobility (EM) and banding phenotypes of isozymes of one PER locus were identical to those of one SOD locus in vegetative buds of five Populus species and hybrid. In leaves of the four Populus species and hybrid and progenies of controlled crosses, EM and phenotypes of isozymes of two PER loci were identical to those of two SOD loci. In Picea species, EM of isozymes of the only SOD locus was somewhat similar but not identical to that of one PER locus, and isozyme phenotypes of all individuals at the SOD locus were not identical to those at a PER locus. Chi-square tests verified the single-gene Mendelian control of the segregating allozyme variants at each of Per-L1 and Sod-1 in the three Populus species. The results of joint two-locus segregation tests indicated a very tight linkage and no recombination between Per-L1 and Sod-1 in three Populus species. Genes coding for isozymes of one or two PER loci are either presumably the same as, or very tightly linked to, the genes coding for isozymes of one or two SOD loci in the Populus species.     

Three amino acid substitutions contributing to thermostability of phosphoglucose isomerase in the Glanville fritillary butterfly

Temperature is one of the most important environmental factors that affect organisms, especially ectotherms, due to its effects on protein stability. Understanding the general rules that govern thermostability changes in proteins to adapt high-temperature environments is crucial. Here, we report the amino acid substitutions of phosphoglucose isomerase (PGI) related to thermostability in the Glanville fritillary butterfly (Melitaea cinxia, Lepidoptera: Nymphalidae). The PGI encoded by the most common allele in M. cinxia in the Chinese population (G3-PGI), which is more thermal tolerant, is more stable under heat stress than that in the Finnish population (D1-PGI). There are 5 amino acid substitutions between G3-PGI and D1-PGI. Site-directed mutagenesis revealed that the combination of amino acid substitutions of H35Q, M49T, and I64V may increase PGI thermostability. These substitutions alter the 3D structure to increase the interaction between 2 monomers of PGI. Through molecular dynamics simulations, it was found that the amino acid at site 421 is more stable in G3-PGI, confining the motion of the α-helix 420–441 and stabilizing the interaction between 2 PGI monomers. The strategy for high-temperature adaptation through these 3 amino acid substitutions is also adopted by other butterfly species (Boloria eunomia, Aglais urticae, Colias erate, and Polycaena lua) concurrent with M. cinxia in the Tianshan Mountains of China, i.e., convergent evolution in butterflies.     

Studies on the properties and tissue distribution of the isozymes of guanylate kinase in man.

The isozyme patterns of guanylate kinase were examined in fetal and adult tissues, in cultured cells and also in red cells separated by density gradient fractionation. Results from fetal and cultured cells inidcated that there are three primary isozymes a, c, and e among the seven isozymes of guanylate kinase in man. Serial secondary isozyme production in red cells in vivo showed that isozyme a produces b, c produces d, and e produces f and g. The three sets of isozymes were found to differ in the following properties: activation/inhibition by EDTA; thermostability, and molecular weights. Isoelectric points of several of the isozymes were estimated by isoelectric focusing. It was concluded that the isozymes of guanylate kinase are determined by three separate gene loci.     

Comparative histochemical studies of iron absorption in three fresh water teleosts.

Absorption of iron in the intestine of three different species of fishes, namely; Claris batrachus, Channa striatus and Esomus danricus has been described. These three species of fishes have different feeding habits, being omnivorus, carnivorus and herbivorus respectively. The absorption of iron has been studied at different time intervals; after three hours, six hours and nine hours. The mucosal epithelial cells, submucosal blood vessels and blood capilaries were the main site of iron absorption. A comparative account of absorption of iron of all three fishes is described and discussed.     

藏北3种裸鲤同工酶的电泳分析及物种分化的探讨

对藏北高原３种裸鲤的乳酸脱氢酶（ＬＤＨ）、苹果酸脱氢酶（ＭＤＨ）和脂酶（ＥＳＴ）进行电泳分析的结果表明,３种裸鲤酶谱均表现出种间的差别,而且在同一种群个体之间也存在着明显的分化,但无性别差异。３种裸鲤被检测的３种同工酶均有沉默基因表达的现象,重复基因ＬＤＨ－Ａ^2、ＬＤＨ－Ｂ^2、s-MDH-A^2和m-MDH-B^2也在部分个体中表达。遗传距离分析表明,色林错裸鲤（Ｇ.selincuoensis)与错鄂裸鲤（Ｇ．cuoensis)之间较之于与纳木错裸鲤（Ｇ．namensis)有更近的亲缘关系。与其他四倍体鱼类相比,裸鲤鱼类同工酶在重复基因和沉默基因上都有较高的表达频率,这种情况说明裸鲤鱼类目前可能还外于多倍化后进化的早期过程并早于胭脂鱼类所处的相应时期,这与裂腹鱼类起源较晚以及青藏高原业已存在的恶劣环境条件直接相关。     

Duplicated phosphoglucose isomerase genes in avocado

Summary Avocado (Persea americana) cultivars were assayed for phosphoglucose isomerase (PGI) isozymes using starch gel electrophoresis. Three PGI genes were identified: one monomorphic locus, Pgi-I, coding for the plastid isozyme and two independently assorting loci, Pgi-2 and Pgi-3, coding for the cytosolic isozymes. The genetic analysis was based on comparisons of PGI zymograms from somatic and pollen tissue and on Mendelian analysis of progeny from selfed trees. The isozymic variability for PGI can be used for cultivar identification and for differentiating between hybrid and selfed progeny in avocado breeding.     

Genetics and polymorphism of a duplicated malate dehydrogenase (MDH) locus in the grasshopperOxya japonica japonica (Orthoptera:Acrididae:Oxyinae)

A biochemical genetic study of the enzyme malate dehydrogenase (MDH) was conducted in the grasshopperOxya j. japonica. Analysis of MDH electrophoretic variation in this species of grasshopper shows that one of the two autosomal loci for MDH in grasshoppers, the Mdh-2 locus, controlling the anodal set of MDH isozymes, is duplicated. Results of breeding studies confirm this and the observed polymorphism at theMdh-2 locus in the two populations ofOxya j. japonica studied can be attributed to three forms of linked alleles at the duplicated locus in equilibrium in both populations. In this respect, all individuals of this species possess heterozygous allelic combinations at the duplicatedMdh-2 locus, which may account for the spread of the duplicated locus in the populations of this species of grasshopper.This research was supported by a grant (Vote F) from the University of Malaya, Kuala Lumpur.     

Glucose 6-phosphate dehydrogenase from brook,lake, and splake trout: An isozymic and immunological study

There are five G6P-specific G6PD isozymes in both brook and lake trout. The most anodal isozyme in each species has the same electrophoretic mobility; however, the five lake trout isozymes are more widely spaced on polyacrylamide gels than are those from brook trout. In hybrids, i.e., splake trout, nine forms of G6PD can be resolved. These results can be explained by a model in which we assume that each isozyme is a tetramer and that two different subunit types are produced. In splake trout, three electrophoretically distinct subunits yield 15 tetramers. That only nine are detected is a consequence of coincident electrophoretic mobility of some of the possible subunit combinations. Our results indicate that the G6PD isozymes in these trout are the products of two codominant autosomal gene loci. The hypothesis that G6PD and H6PD arose from a common ancestral type G6PD is supported by microcomplement fixation data which show an immunochemical relatedness between these enzymes. The relationship of G6PD and H6PD in trout is discussed from an evolutionary standpoint.This research was supported in part by National Science Foundation Grant GB-7271 and by United States Public Health Predoctoral Fellowship 4 FO1 GM 43657-03.