Expression of lactate dehydrogenase phenotypes in intraspecific and interspecific matings of two species of Xenopus

Starch gel electrophoresis has been used to examine lactate dehydrogenase phenotypes in two species of Xenopus and their hybrids obtained from reciprocal crosses. The patterns are complex, consisting of as many as 18 bands in some material. Differences between laevis and mulleri isozymes allow an evaluation of the contribution of both parents to the phenotypes of their hybrid offspring, and the determination of approximate times of paternal allele expression. The phenotype of early embryos resembles that of the maternal parent until hatching, when evidence of paternal influence is first apparent. Regardless of the early appearance of paternal enzyme, reciprocal hybrids bear a stronger resemblance to the maternal parent until well after tadpole growth begins. Once this maternal effect disappears, both laevis and mulleri appear to contribute to the LDH phenotype without predominance of the isozymes of either species. Evidence for the possible formation of “hybrid” enzymes consisting of subunits of both species in one active enzyme molecule is presented. Expression of LDH phenotype is variable in the unfertilized eggs of fertile hybrid females.     

Lactate dehydrogenase ontogeny,paternal gene activation,and tetramer assembly in embryos of brook trout,lake trout,and their hybrids

Measurement of lactate dehydrogenase in reciprocal hybrids of trout during development revealed that a maternal effect was involved in the regulation of enzyme levels until resorption of the yolk sac was completed. Malate dehydrogenase specific activities were the same in these embryos and larvae. The more negatively charged B subunits of LDH predominated during early stages of embryogenesis in lake trout and brook trout with an increase in synthesis of A subunits evident as development progressed. Activation of the paternal A gene in reciprocal hybrids occurred at a relatively late stage with the LDH subunit specific to the retina appearing after hatching. Analysis of brook trout progeny from a cross of parental types with a variant and wild-type B subunit suggested nonrandom LDH tetramer assembly which may be genetically controlled.This study was supported by National Science Foundation Grant GB-7271.     

The ontogeny of phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in Japanese quails and chicken-quail hybrids

The ontogeny of phosphogluconate dehydrogenase in Japanese quails and chicken-quail hybrids was determined by vertical starch gel electrophoresis of pooled early embryos. The enzyme of maternal origin was found to be present at all stages of development. Paternal phosphogluconate dehydrogenase in pure quails was detected as early as the fourth hour of incubation, whereas that in chicken-quail hybrids was not observed until the eighth hour. Since embryonic development up to the day of hatching was generally retarded in chicken-quail hybrids when compared to pure quails, it was concluded that the embryonic gene coding for phosphogluconate dehydrogenase was expressed at the same stage of development in both quails and hybrids. The ontogeny of glucose 6-phosphate dehydrogenase in chicken-quail hybrids was compared to that of phosphogluconate dehydrogenase. The paternal form of both enzymes first appeared at the eighth hour of incubation, indicating that these two functionally related genes coding for the corresponding enzymes were probably activated at the same time in development.     

Ontogeny of lactate dehydrogenase isozymes in chicken-quail hybrid embryos

The ontogeny of lactate dehydrogenase (LDH) isozymes was examined in avian hybrids and compared with the isozyme patterns of the parental species. Hybrids were obtained by crossing female Japanese quali (Coturnix coturnix japonica) with male domestic chickens (Gallus gallus domesticus). By use of starch gel electrophoresis and an enzyme-specific stain, traces of embryonic paternally derived LDH were detected in unincubated hybrid eggs. It was concluded that the embryonic genes coding for the B subunits of LDH are activated during the hours between fertilization and oviposition. In early blastoderms, a great excess of maternally stored LDH is present. In the hybrid, the predominantly maternal pattern of isozymes shifts during embryogenesis to a predominantly paternal pattern. This was considered evidence for differential allelic regulation of LDH inactivation. A progressive trend toward the establishment of the adult distribution of isozymes in various tissues was also observed in the hybrid and quail, and found to be similar to chicken LDH isozyme ontogeny.     

Gene activation of alcohol dehydrogenase in danio hybrids

The regulation of alleles encoding the enzyme alcohol dehydrogenase (ADH) was investigated in F1Brachydanio hybrids (zebra danio female x spotted danio male) by acrylamide gel electrophoresis. Both parental species showed a single, cathodal band of species-specific ADH. During development at 26 degrees C, hybrid fry showed a preferential activation of the maternally derived Adh allele. It is suggested that the low activity of the paternally derived allele may result from an incompatibility between maternal regulatory factors and the paternal regulative element controlling gene expression.     

In vitro effects of polyvinylpyrrolidone and sucrose on the acetylcholinesterase,succinate dehydrogenase and lactate dehydrogenase activities in the brain

Summary The supernatant prepared from the brain tissue homogenate incubated in vitro in the presence of PVP or sucrose exhibits a decrease of AChE, SDH as well as of LDH activity. A 0.75% PVP solution inhibits AChE activity by 30%, LDH activity is inhibited by 35% and SDH activity by 40%. A two hours lasting effect of a 7.5% PVP solution at 3° C on enzymatic preparations induces in AChE 20% inhibition of its activity, in LDH an inhibition of 44% and in SDH the inhibition of its activity amounts to 74%. 1 M Sucrose inhibits AChE activity by 34%, LDH activity by 41% and SDH activity is inhibited by 31%. After two hours lasting effect of 1.4 M sucrose at 3° C on the supernatant the AChE activity is inhibited by 22% and that of LDH by 30%. The SDH activity was after a two hours lasting effect of 1 M sucrose at 3° C inhibited by 34%. The inhibition of activity of the above mentioned enzymes localized in brain cortex preparations was compared with the inhibition of activity of the isolated serum cholinesterase. 0.25 M Sucrose inhibited the activity of this enzyme by 25% and 0.75% PVP by 45%. A two hours lasting effect of 7.5% PVP or 1 M sucrose at 3° C on the cholinesterase induced a 40% and 22% inhibition respectively. After double washing of the brain cortical minced tissue, prepared in a 7.5% PVP containing solution, AChE activity was constant. By triple washing of the brain cortical crude mitochondrial fraction, exposed for two hours at 3° C to the effect of 1 M sucrose, SDH activity was also constant.Abbreviations AChE acetylcholinesterase (EC 3.1.1.7.) - INT 2(p-iodophenyl)3-p-nitrophenyl-5-phenyl tetrazolium chloride - LDH lactate dehydrogenase (EC 1.1.1.27.) - PMS phenazine methosulfate - PVP polyvinylpyrrolidone - SDH succinate dehydrogenase (EC 1.3.99.1.)     

Heat stability of cholinesterase and non-specific esterases during development of hybrids of the sea urchins Stongylocentrotus droebachiensis and S. intermedius]

Acetylcholine esterase (AchE) and non-specific esterases were studied during the development of sea urchins Strongylocentrotus droebachiensis and S. intermedius and their hybrids by means of electrophoresis and measurements of enzyme thermostability. Two AchE fractions were found which differed by thermostability. At the late gastrula stage, the therolabile form predominated and at the mid-pluteus stage the thermostable one. Non-specific esterases in both the species of sea urchins are represented by complex isozyme systems. Their changes during development are accompanied by the changes in thermostability and electrophoretic patterns. The thermostability of esterases at the pluteus stage in the hybrids is higher than in the maternal species, apparently, due to the appearance of the thermostable enzyme which appears in the paternal species provisionally at the prism stage.     

Allelic expression at the sorbitol dehydrogenase and glucosephosphate isomerase loci in an interspecific hybrid ricefish

1. The expression of alleles encoding the enzymes sorbitol dehydrogenase (SDH; EC 1.1.1.14) and glucosephosphate isomerase (GPI; EC 5.3.1.9) was investigated in Oryzias melastigma, O. javanicus and in O. melastigma female x O. javanicus male hybrids by acrylamide gel electrophoresis. 2. It was not possible to investigate the expression of SDH or GPI in reciprocal hybrids as these fry failed to develop past the stage of embryonic body formation. 3. The delay in appearance of isozymes of paternal SDH subunit composition, and paternal and maternal GPI-B subunit composition is in keeping with observed effects of gene regulatory divergence where alleles of maternal origin interact more effectively with maternal cytoplasmic regulatory factors than do alleles of paternal origin.     

Asynchronous expression of the alcohol and supernatant malate dehydrogenase loci during Barbus hybrid development (Cypriniformes, Teleostei)

The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) and alcohol dehydrogenase (ADH) are reported for the tiger barb (Barbus tetrazona), the rosy barb (Barbus conchonius) and their reciprocal hybrids. The tissue distribution of s-MDH and ADH isozymes in both species is consistent with spatial profiles reported for other teleosts. The expression of alleles of paternal origin at the s-Mdh-B and Adh loci are delayed in reciprocal hybrids as compared to their expression intraspecifically; suggestive of a low degree of affinity between maternally derived regulatory factors and paternal regulative elements controlling structural gene activation.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

Intracellular Location and Properties of Plant L-Glutamate Dehydrogenases

A study of some properties of L-glutamate dehydrogenase (GDH)in the supernatant and mitochondrial fractions of mung beanhypocotyls failed to reveal any differences between the twoenzymes. GDH in lettuce leaf chloroplasts was solubilized withTriton-X-100 and marked differences in a number of propertieswere found between this enzyme and the one solubilized fromlettuce leaf mitochondria. It was concluded that the mitochondrialand chloroplastic GDH's are distinct enzymes. The small amountof GDH activity detected in isolated lettuce leaf peroxisomescould have been due to adsorption of the enzyme.     

Molecular gene cloning, expression, and characterization of bovine brain glutamate dehydrogenase

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The Km and Vmax values for NAD+ were 0.1 mM and 1.08 micromol/min/mg, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - 100 microM, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.     

The ontogenesis of catabolic abilities and energy metabolism during endogenous nutritional periods of tongue sole,Cynoglossus semilaevis

The ontogenesis of catabolic abilities and energy metabolism during endogenous nutritional periods of tongue sole was investigated. In this work, trypsin-like proteases (TRY) and triglyceride lipase (LIP) activities were measured to assess the capacities to catabolize proteins and lipids, respectively. Meanwhile, specific enzymes including pyruvate kinase (PK), glutamic oxalo acetic transaminase (GOT) and glutamate dehydrogenase (GDH), and hydroxyacyl CoA dehydrogenase (HOAD) as well as their ratios were assayed to evaluate the abilities to use energy substrates of carbohydrates, amino acids and fatty acids, respectively, for energy production. In addition, activities of citrate synthase (CS) and lactate dehydrogenase (LDH) and LDH/CS ratio were calculated to analyse the evolution of aerobic and anaerobic pathways. The study found that hatching occurred at 38.8 h after fertilization (HAF), mouth-opening day of eleuteroembryo appeared at 3 days after hatching (DAH), and the most rapid embryonic growth was observed in blastula stage before hatching. Enzymatic assay revealed that except for PK which appeared in cleavage stage onwards, all the other enzymes functioned after fertilization, preparing well for the coming embryogenesis of tongue sole. By comparing the average specific activity of enzyme in each period, it can be found that the highest value occurred at 3 DAH (for TRY, LIP, PK and LDH), 2 DAH (for GDH), fertilized egg (for GOT) and segmentation stage (for HOAD and CS), and the lowest value occurred at fertilized egg (for HOAD, CS and GDH), cleavage stage (for TRY, PK and LDH), gastrula stage (for GOT) and hatching day (for LIP). Based on the changeable patterns of metabolic enzymatic activities and ratios, it is concluded that metabolic capacities on three energy substrates displayed stage-specific traits, and the dominant energy substrate was fatty acids before segmentation stage, amino acids until hatching day and carbohydrate during eleuteroembryo period. As for energy production mode, aerobic pathway appeared to increase greater in fertilized egg and gastrula stage, whereas anaerobic pathway played a predominant role during cleavage stage, blastula stage, segmentation stage and eleuteroembryo stage. These results are valuable to elucidate the nutritional requirements of embryonic stages in tongue sole and to further understand their energy metabolic mechanisms.     

酶的基因剂量效应及其对鱼类远缘杂交的影响

亲缘关系较远的杂种二倍体,酶的表达出现父本基因受抑制,双亲基因表达迟缓和母本基因提前表达三种类型的紊乱。由于双亲等位基因不亲和,各等位基因只调控产生1／2的基因产物,从而致使胚胎出现“双单倍体综合症”而在孵化期全部死亡。远缘杂交三倍体由于母本基因剂量加倍,一方面抑制了父本基因的表达,消除了不协调基因活动的干扰,另一方面母本基因产物趋于正常,代谢得以正常进行,杂种后代能成活并正常生长。     

Comparison of the spatial and temporal expression of supernatant malate dehydrogenase in Barbus hybrids (Cypriniformes, Teleostei)

The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) are reported for the tiger barb, cherry barb, and their reciprocal hybrids. The tissue distribution of s-MDH isozymes in Barbus is consistent with the patterns reported in other teleosts. The expression of the Mdh-B locus is correlated with the initial muscle contractions of the developing embryos. It is suggested that the state of muscle cell differentiation may be the stimulus necessary for the expression of this locus in Barbus. Expression of maternal and paternal alleles at the B locus are synchronously delayed in reciprocal hybrids, as compared to their expression intraspecifically.     

A thermally sensitive loop in clostridial glutamate dehydrogenase detected by limited proteolysis

The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C. Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site. SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa. Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change. Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH. Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme. Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments. However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure. Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states. N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.     

Enzymatic urea adaptation: lactate and malate dehydrogenase in elasmobranchs

Lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) electrophoretic tissue patterns of two different orders of Elasmobranchii: Carchariniformes (Galeus melanostomus and Prionace glauca) and Squaliformes (Etmopterus spinax and Scymnorinus licha) were studied. The number of loci expressed for these enzymes was the same of other elasmobranch species. Differences in tissue distribution were noted in LDH from G. melanostomus due to the presence of an additional heterotetramer in the eye tissue. There were also differences in MDH. In fact, all the tissues of E. spinax and G. melanostomus showed two mitochondrial bands. Major differences were noted in the number of isozymes detected in the four compared elasmobranchs. The highest polymorphism was observed in E. spinax and G. melanostomus, two species that live in changeable environmental conditions. The resistance of isozymes after urea treatment was examined; the resulting patterns showed a quite good resistance of the enzymes, higher for LDH than MDH, also at urea concentration much greater than physiological one. These results indicated that the total isozyme resistance can be considered higher in urea accumulators (such as elasmobranchs) than in the non-accumulators (such as teleosts).     

Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies

Somatic hybrids of drug-resistant mutant hamster and mouse cell lines have been isolated and propagated in long-term culture and have been studied in respect to karyotype and three enzymes. During the course of propagation the long-surviving hybrid clones show progressive loss of telocentric chromosomes associated in at least one case with loss of mouse enzyme. Hybrid clones showed hybrid molecules for malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6PGD) made up by recombination of parental subunits.This work was supported by National Institutes of Health Grant HD 00486.     

Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs

Glutamate dehydrogenases (GDHs) from fresh-water and marine hyperthermophilic Archaea were compared with respect to their responses to different salt concentrations. A gene encoding GDH from the terrestrial hyperthermophilic archaeon Thermococcus waiotapuensis (Twaio) was cloned, sequenced, and expressed at a high level in Escherichia coli. The deduced amino acid sequence, which consists of 418 amino acid residues, revealed a high degree of similarity with GDHs from related marine strains such as Thermococcus litoralis (Tl) and Pyrococcus furiosus (Pfu). Recombinant Twaio GDH was purified 27-fold to homogeneity. The enzyme is hexameric with a molecular weight of 259,000. The effects of several salts (KCl, CaCl, MgSO4), temperature, and pH on enzyme activity were determined and compared in three hyperthermophilic GDHs, including T. waiotapuensis, and GDHs from two marine species, T. litoralis and P. furiosus. Kinetic studies suggested a biosynthetic role for the nicotinamide adenine dinucleotide phosphate- (NADP-) specific Twaio GDH in the cell. Interestingly, Twaio GDH revealed no salt responses, whereas the two marine GDHs showed substantial enhancement of activity as well as thermostability at increasing salt concentrations. Because electrostatic interactions between charged amino acid residues are thought to be a key feature of structural integrity and thermostability in hyperthermophilic GDHs, salt availability and its effects on marine enzymes could partially explain a higher thermal stability in marine species than in phyletically related fresh-water species.     

Expression and in vitro assembly of recombinant glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

The gdhA gene, encoding the hexameric glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus furiosus, was expressed in Escherichia coli by using the pET11-d system. The recombinant GDH was soluble and constituted 15% of the E. coli cell extract. The N-terminal amino acid sequence of the recombinant protein was identical to the sequence of the P. furiosus enzyme, except for the presence of an initial methionine which was absent from the enzyme purified from P. furiosus. By molecular exclusion chromatography we showed that the recombinant GDH was composed of equal amounts of monomeric and hexameric forms. Heat treatment of the recombinant protein triggered in vitro assembly of inactive monomers into hexamers, resulting in increased GDH activity. The specific activity of the recombinant enzyme, purified by heat treatment and affinity chromatography, was equivalent to that of the native enzyme from P. furiosus. The recombinant GDH displayed a slightly lower level of thermostability, with a half-life of 8 h at 100 degrees C, compared with 10.5 h for the enzyme purified from P. furiosus.