The effect of temperature acclimation on NADP-dependent dehydrogenase activity in the carp liver and various mechanisms of its regulation

Acclimation of carp both to the temperature fall (from 20 to 5 degrees C) and rise (from 20 to 30 degrees C) induces an increase in activity of cytoplasmic liver NADPH-generating enzymes--glucose-6-phosphate dehydrogenase (G6PDG) and malic-enzyme (ME) 6-phosphogluconate dehydrogenase (6PGDG) and NADP-isocitrate dehydrogenase (NADP-IDG) activities are unchanged. Actinomycin D does not prevent cold activation of G6PDG but blocks activation of ME. "Warm" G6PDG has minimal Km value for glucose-6-phosphate and "warm" ME has minimal Km value for glucose-6-phosphate and "warm" ME has minimal Km value for malate at 25 degrees C "Cold" G6PDG and ME have the warmest Km values at 5 degrees C. Isozyme composition of cytoplasmic G6PDG (2 bands with Rf 0.16 and 0.20) does not change within the limits of 5-30 degrees C. The prolactin action on G6PDG and ME is similar to the effect of cold acclimation (activity increases Km value decreases, isozyme pattern (for G6PDG) remains unchanged). It is supposed that activation of G6PDG and ME during cold adaptation may be a result of the prolactin action on substrate-binding ability without changes in the enzyme biosynthesis and isozyme pattern.     

TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响

对酿酒酵母在添加苹果酸、柠檬酸和琥珀酸的混合培养基与其在YEPD培养基中胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活力差异进行了对比分析。结果表明:添加苹果酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.82%、57.23%、39.15%、12.10%;添加柠檬酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶的酶活分别下降50.17%、42.20%、48.40%;添加琥珀酸使胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.16%、34.16%、50.87%、50.87%、12.37%。丙酮酸激酶、异柠檬酸脱氢酶和苹果酸脱氢酶对3种有机酸的耐受性较差,葡萄糖-6-磷酸脱氢酶、乙醇脱氢酶对3种有机酸的耐受具有选择性。     

Sex differences in the control of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Interaction of estrogen, testosterone and insulin in the regulation of enzyme levels in vivo and in cultured hepatocytes

Control of the activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malate dehydrogenase was investigated in intact rats and in hepatocyte cultures. 1) Adult females had 2-fold greater activities of hepatic glucose-6-phosphate- and 6-phosphogluconate dehydrogenases than adult males, but similar activities of malate dehydrogenase. Castrated males showed decreased activities of all three enzymes in comparison to age- and weight-matched intact controls. In starved animals the activities of all three enzymes decreased significantly. After refeeding with nonpurified diet the activities returned to the prestarved levels in females, but increased to clearly higher values in intact and castrated males. 2) Estrogen levels were in the same range in immature and adult male and female rats. Testosterone levels were highest in adult males, clearly lower in adult females (1/8) and immature males (1/8), still lower in immature females (1/15) and lowest in castrated males (1/40). A simple correlation of the sex differences in these hormone levels to sex differences in glucose-6-phosphate- and 6-phosphogluconate dehydrogenase activities was not apparent. 3) In serum-free, dexamethasone-supplemented 48-h cultures of hepatocytes from both male and female rats the basal activities of glucose-6-phosphate dehydrogenase were the same; they were increased 2-3 fold by insulin alone, 1.5 fold by estrogen alone and 4-5 fold by insulin plus estrogen. Apparently sex differences did not persist in 48-h cell cultures. 4) In 48-h cultures of male hepatocytes, then used as the experimental model, insulin alone increased the activity not only of glucose-6-phosphate dehydrogenase but also of 6-phosphogluconate and malate dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium dodecyl sulfate, concurrent inhibitor of several dehydrogenases

The effect of sodium dodecyl sulfate on the activity of highly purified or crystalline enzymes has been studied. The enzymes were: lactate dehydrogenase (LDH), malate dehydrogenase (MDH). isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6P-DH), lipase, alkaline phosphatase. Sodium dodecyl sulfate, always under the critical micellar concentration, shows a selective inhibitory effect. A kinetic analysis of the inhibitory action on LDH, MDH, ICDH and G6P-DH was also carried out.     

Affinity electrophoresis of proteins interacting with Blue dextran.

Interaction of several enzymes (pyruvate kinase, myokinase, creatine kinase, aldolase, malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase) and other proteins (bovine serum albumin and ovalbumin) with Blue Dextran was studied by means of affinity electrophoresis in polyacrylamide gels. A decrease of electrophoretic mobility of enzymes in affinity gels was dependent on Blue Dextran concentration and in some cases, dissociation constants of the protein-immobilized dye complexes could be calculated. Affinity electrophoresis in the presence of Blue Dextran reveals in some cases additional bands of isoenzymes, as compared with the control gels (without Blue Dextran).     

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans

Abstract Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10–11) for up to 6 h and 3 h at 25°C, respectively, while at pH 6.5, 50% of their activities were lost within 2–3 h. The K _m for malate oxidation catalysed by MDH was 5.8×10⁻⁴ M while that for glucose-6-phosphate oxidation was 2.0×10⁻⁴ M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.     

Regulation of Crassula argentea phosphoenolpyruvate carboxylase in relation to temperature.

The effect of temperature on the kinetic parameters of phosphoenolpyruvate carboxylase purified from Crassula argentea was such that both the Vmax and Km(MgPEP) values tended upward over the range from 11 to 35 degrees C. The increased rate at low temperatures due to the low Km is at least partially offset by the increased Vmax at higher temperatures, potentially leading to a broad plateau of enzyme activity and a relatively small effect of temperature on the enzyme. The cooperativity was negative at 11 degrees C, but above 15 degrees C it became positive. The presence of 5 mM glucose-6-phosphate has relatively little effect on Vmax but it clearly reduces Km and overcomes any effect of temperature on this parameter in the range studied. Positive cooperativity is observed only at temperatures above 25 degrees C. The size of the native enzyme, as determined by dynamic light scattering, was strongly toward the tetrameric form. At a temperature of 40 degrees C and above, a considerable oligomerization takes place. No loss of activity can be observed in this range of temperature. In the presence of either glucose-6-phosphate or magnesium phosphoenolpyruvate, at temperatures under 25 degrees C, the equilibrium is displaced toward higher levels of aggregation. Maximal accumulation of lead malate occurred at 10 to 12 degrees C in vivo with reduction to about 25% at 35 degrees C. Glucose-6-phosphate followed a similar curve in response to temperature, but the overall difference was about 50%. The sum of phosphoenolpyruvate plus pyruvate is level at night temperatures below 25 degrees C, doubling at 35 degrees C. Calculated concentrations of malate, glucose-6-phosphate, and phosphoenolpyruvate plus pyruvate indicate that the concentrations present are equal to or greater than Ki, Ka, and Km values for these metabolites, respectively.     

Plant Leaf and Stem Proteins. II. Isozymes and Environmental Cabbage

The activity of 10 enzymes separated by acrylamide disc gel electrophoresis of leaf and stem extracts from Dianthus grown under summer and winter conditions was studied. While banding was constant and highly reproducible under each environment, differences between the 3 cultivars and between the tissues were evident. No significant differences in the isozyme patterns of glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and catalase were observed between the 2 environments. Loss of activity was observed under winter conditions with amylase and lactate dehydrogenase and loss of certain isozymic components was evident with acid phosphatase and esterase. Prominent changes were observed in peroxidase isozymes, the hardy cultivars developing additional isozymic components under winter conditions. Only minor changes in the total protein banding were seen. The enzymes showed considerable stability in those tissues killed by the freezing conditions.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Consequences of the presence of 24-epibrassinolide, on cultures of a diatom, Asterionella formosa

Addition of the plant hormone 24-epibrassinolide to culture media stimulated the growth of a freshwater diatom, Asterionella formosa. The hormone stimulated activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme from Calvin cycle, by 6-fold. Other key metabolic enzymes, phosphofructokinase and malate dehydrogenase were also stimulated but to a lesser extent. The activity of glucose-6-phosphate dehydrogenase, involved in the oxidative pentose phosphate pathway, also increased in the presence of the hormone but only under non reducing conditions. In cells stimulated by epibrassinolide, activated enzymes were sensitive to oxidized-DTT. GAPDH purified from cells grown in the presence of the hormone was not associated with a small protein of 8.5 kDa shown to be similar to CP12. Consequently the activity of GAPDH was no longer regulated by either oxidizing or reducing conditions. Among enzymes that, like GAPDH, responded positively to reducing agent were fructose-1,6-bisphosphatase (FBPase) and glucose-6-phosphate dehydrogenase (G6PDH). These enzymes were also sensitive to, and were negatively regulated by, oxidized-DTT. The activities in extracts from illuminated cells differed from those from darkened cells: FBPase, G6PDH and GAPDH, that were activated by DTT in darkened cells were no more activated in illuminated cells, but were oxidized by oxidized-DTT. Thus, oxidizing or reducing conditions mimic the conditions in dark and light, respectively. Unlike the other enzymes, phosphofructokinase (PFK) was inhibited by DTT but oxidized-DTT reversed this effect. The enzymes shown to be redox regulated in vitro by reduction/oxidation are very likely candidates for regulation in vivo by thioredoxins.     

Metabolic background for establishment of the subpopulation structure in early ontogenesis in the Atlantic salmon (the case of energy of carbohydrate metabolism)

The activity of some enzymes involved in energy and carbohydrate metabolism was studied in Atlantic salmon embryos at the eyed egg stage and in salmon fingerlings (0+) from two trophic–ecological groups: the Varzuga River bed and two tributaries, the Pyatka and Sobachii rivers (Kola Peninsula). It has been demonstrated that heterogeneity of embryos was most evident in the case of cytochrome c oxidase (CO), malate dehydrogenase (MDH), glycerol-1-phosphate dehydrogenase (G1PDH), and glucose-6-phosphate dehydrogenase (G6PDH), while the lowest level of heterogeneity was observed for lactate dehydrogenase (LDH) and aldolase. A positive correlation was revealed between the activities of CO, LDH, MDH, and G1PDH. It was noted that G6PDH showed a negative correlation with almost all enzymes under study. It was found that salmon juveniles inhabiting the tributaries were characterized by high LDH, aldolase, and G1PDH activity and lower activity of G6PDH compared to the juveniles inhabiting the main river bed. Notably, the differences in the activity of the enzymes involved in aerobic metabolism between the two groups of fingerlings under analysis were observed only in the autumn.     

Isoenzymes in two micromycetes species: taxonomic aspects

Strains of different origins belonging to the two species of fungi imperfecti Cylindrocarpon ianothele and Calcarisporium arbuscula exhibit difference in their morphological or physiological appearances. We have attempted to determine if these variabilities could also be observed at the level of other phenotypic markers, the enzymes glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, octanol dehydrogenase and malate dehydrogenase. Isozymes were separated by polyacrylamide gel electrophoresis and demonstrated isozyme variability as a function of growth and physiological states of a given strain. At the same state of development, the different strains exhibited highly heterogeneous isozyme groups which appear difficult to use for taxonomic purposes.     

A comparison of isozymes of five axenic Giardia isolates

The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.     

Allozyme comparison of three Trypanosoma species (Kinetoplastida: Trypanosomatidae) of toads and frogs by starch-gel electrophoresis.

Six metabolic enzymes, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, isocitrate dehydrogenase, malate dehydrogenase, phosphoglucomutase, and purine nucleoside phosphorylase, from clonal isolates of 3 presumptive species of Trypanosoma (T. fallisi, T. ranarum, and T. rotatorium) from 3 anuran hosts (Bufo americanus, Rana clamitans, and Rana catesbeiana) were compared using starch-gel electrophoresis. Although bands were shared among the different zymodemes of isolates of the same host genus, low genetic polymorphism of the enzyme loci was observed with few apparent shared bands between samples isolated from frogs and toads. A distance value calculated between toad and frog trypanosome isolates suggests the likelihood of long-time separation of species. Cluster analysis based on overall similarity distinguished the trypanosomes of toads and frogs as separate taxa, suggesting that host specificity and observed morphological differences are consistent with heritable allozyme differences.     

Enzymes of Energy Metabolism in the Mudpuppy Retina

Abstract: The distributions of glycogen phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, citrate synthase, malate dehydrogenase, β-hydroxyacyl CoA dehydrogenase, and adenylokinase were determined in the mudpuppy retina. Distinct differences were found in regard to the glycolytic and oxidative capacities of the various layers. In the outer retina, citric acid cycle enzymes were high while glycolytic enzymes were low. Synaptic zones were distinctly enriched in all energy-producing enzymes. Mudpuppy photoreceptors were found to be rich in phosphorylase but poor in glucose-6-phosphate dehydrogenase, suggestive of some evolutionary divergence from mammals in the metabolic machinery which is used to support the visual process.     

Histochemical observations on the exoerythrocytic malaria parasite Plasmodium berghei in rat liver

Enzyme histochemical methods were performed on sporozoite infected liver tissue of rats in order to gain insight into the nutrition and metabolism of exoerythrocytic forms of Plasmodium berghei. The following enzymes were demonstrated in the hepatocytic stages of the parasites, obtained 41 and 48 h after inoculation of sporozoites: acid phosphatase, cytochrome oxidase, NADH-tetrazolium reductase, succinate dehydrogenase, NAD+ and NADP+ dependent isocitrate dehydrogenase, NADP+-dependent malate dehydrogenase, lactate dehydrogenases, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenases and alpha-glycerol-phosphate dehydrogenase. The results suggest that a conventional Embden-Meyerhoff pathway, pentose phosphate pathway and Krebs' citric acid cycle may in part be present in these exoerythrocytic parasites. Alkaline phosphatase, nucleoside polyphosphatase, 5' nucleotidase, glucose-6-phosphatase, alpha-glucan phosphorylase, NAD+ dependent malate dehydrogenase, amino-peptidase M and non-specific esterases were not detected by our techniques in the parasite. The enzyme distribution of this intrahepatocytic malaria parasite revealed by histochemistry is compared with the enzyme distribution in the other phases of the parasite's life cycle.     

Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.     

Oxidative stress reversibly inactivates myocardial enzymes during cardiac arrest

Oxidative stress during cardiac arrest may inactivate myocardial enzymes and thereby exacerbate ischemic derangements of myocardial metabolism. This study examined the impact of cardiac arrest on left ventricular enzymes. Beagles were subjected to 5 min of cardiac arrest and 5 min of open-chest cardiac compressions (OCCC) before epicardial direct current countershocks were applied to restore sinus rhythm. Glutathione/glutathione disulfide redox state (GSH/GSSG) and a panel of enzyme activities were measured in snap-frozen left ventricle. To test whether oxidative stress during arrest inactivated the enzymes, metabolic (pyruvate) or pharmacological (N-acetyl-l-cysteine) antioxidants were infused intravenously for 30 min before arrest. During cardiac arrest, activities of phosphofructokinase, citrate synthase, aconitase, malate dehydrogenase, creatine kinase, glucose-6-phosphate dehydrogenase, and glutathione reductase fell by 56, 81, 55, 34, 42, 55, and 45%, respectively, coincident with 50% decline in GSH/GSSG. OCCC effected full recovery of glutathione reductase and partial recovery of citrate synthase and aconitase, in parallel with GSH/GSSG. Phosphofructokinase, malate dehydrogenase, creatine kinase, and glucose-6-phosphate dehydrogenase recovered only after cardioversion. Antioxidant pretreatments augmented phosphofructokinase, aconitase, and malate dehydrogenase activities before arrest and enhanced these activities, as well as those of citrate synthase and glucose-6-phosphate dehydrogenase, during arrest. In conclusion, cardiac arrest reversibly inactivates several important myocardial metabolic enzymes. Antioxidant protection of these enzymes implicates oxidative stress as a principal mechanism of enzyme inactivation during arrest.     

Specific interaction among some enzymes and sodium dodecyl sulfate

The effect of 1-butanesulfonic acid sodium salt and sodium dodecyl sulfate on the activity of highly purified and crystalline enzymes with marked differences in structure and function has been studied. The enzymes were: alcohol dehydrogenase; lactate dehydrogenase; malate dehydrogenase; isocitrate dehydrogenase; glucose-6-phosphate dehydrogenase; lipase; alkaline phosphatase. While 1-butanesulfonic acid sodium salt, at the studied concentrations, resulted generally inactive, sodium dedecyl sulfate showed a selective inhibitory effect, always under the critical micellar concentration. A kinetic analysis of the inhibitory action was also carried out.     

Genetic studies of free-ranging macaques of Cayo Santiago. I. Description of the population and some nonpolymorphic red cell enzymes.

Phenotypes of eight red cell enzymes at nine genetic loci were determined in the semi-free-ranging population of rhesus macaques; Macaca mulatta, that inhabit Cayo Santiago. The following enzymes were examined electrophoretically: adenosine deaminase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, indophenol oxidase, lactate dehydrogenase, malate dehydrogenase, phosphoglucomutase-1, phosphoglumutase-2, and purine nucleoside phosphorylase. Hemolysates from at least 372 animals were analyzed, and no variants of the enzymes were observed with the exception of malate dehydrogenase. Three animals displaying a variant form of malate dehydrogenase were found.