Subcellular distribution and properties of aldehyde dehydrogenase in the rat liver

The subcellular distribution and certain properties of rat liver aldehyde dehydrogenase are investigated. The enzyme is shown to be localized in fractions of mitochondria and microsomes. Optimal conditions are chosen for detecting the aldehyde dehydrogenase activity in the mentioned fractions. The enzyme of mitochondrial fraction shows the activity at low (0,03-0.05 mM; isoenzyme I) and high (5 mM; isoenzyme II) concentrations of the substrate. The seeming Km and V of aldehyde dehydrogenase from fractions of mitochondria and microsomes of rat liver are calculated, the acetaldehyde and NAD+ reaction being used as a substrate.     

Changes in the activity of alcohol dehydrogenase isoenzymes during the estrus cycle in the vagina of the rat

In rodents, the vaginal epithelium undergoes cyclical changes with an alternating pattern of keratinization and mucification. It has been known for decades that vitamin A and its active form retinoic acid are responsible for normal epithelial homeostasis. However, it has not so far been certain which enzymes catalyze the first and rate-limiting step in retinoic acid synthesis. By means of microdissection and ultrathin-layer gel electrophoresis, alcohol dehydrogenase isoenzyme activity was determined quantitatively in the various layers of the vaginal mucous membrane. It was found that, in the rat, only alcohol dehydrogenase 3 and 4 are expressed. Marked cyclical changes of alcohol dehydrogenase 4 activity in the stratum germinativum of the vaginal epithelium strongly support the assumption that this isoenzyme is responsible for retinoic acid synthesis, and that it is essential for the changes accompanying keratinization and mucification.     

Enzyme Activities in Regions of the Hypothalamus

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].     

Myonic makeup of the muscles innervated by the vagus nerve

Histochemical investigation on succinic dehydrogenase activity and morphometric studies have demonstrated certain differences in the dog sublingual group of muscles. The thyreohyoid and sternohyoid muscles innervated by spinal nerves possess three types of myons differing in succinic dehydrogenase activity and in the area of transversal section. The cricothyreoid muscle and the superior pharyngeal constrictor obtaining their motor innervation from the vagus nerve are composed of unitypical muscular fibres with nearly the same areas of transversal section and high enzymic activity. The differences noted should be explained by different sources of motor innervation.     

A possible contribution by glial cells to neuronal energy production enzyme-histochemical studies in the developing rat cerebellum

Summary Recent reports have revealed that certain neurons do not survive in vitro in the presence of glucose, which is the primary substrate and exclusive source of energy in the brain. But these neurons can survive in the presence of low-molecular-weight agents such as pyruvate, which are supplied by glial cells (Selak et al. 1984). To test whether this result also holds true in vivo, we investigated the distribution of hexokinase, lipoic dehydrogenase, -hydroxybutyrate dehydrogenase, and glucose-6-phosphate dehydrogenase activities in the developing rat cerebellum. Hexokinase activity was relatively higher in glial cells than in neurons. After postnatal day 8, the activity of hexokinase could hardly be detected in Purkinje cells, whereas it was highest in Bergmann glial cells. Purkinje cells were the only type of neuron with high levels of lipoic dehydrogenase at all ages tested. -Hydroxybutylate dehydrogenase activity was also high in Purkinje cells, especially in those from young rats. Relatively high glucose-6-phosphate dehydrogenase activity was demonstrated in basket and stellate cells from adult brain. Thus, it appears that, in vivo, certain neurons utilize relatively little glucose, and it is indeed possible that glial cells may supply some substance(s) other than glucose, for example pyruvate, as the primary source of energy.     

Release of myoglobin in the blood of irradiated swine showing decreased activity of various enzymes--markers of muscle tissue injury

Dynamics of changes in the level of myoglobin, hematological indices, and activity of certain enzymes (lactate dehydrogenase, hydroxybutyrate dehydrogenase, and creatine phosphokinase) was studied in blood of exposed (103.2 mC/kg) pigs. As the activity of enzymes decreased the myoglobin content of blood increased 5-7 days following irradiation. The effect of radiation was shown to promote the development of hypoxia in the animal body which was indicated by the ECG changes, the release of functionally deficient erythrocytes to blood, and the occurrence of stable vast hemorrhages.     

Enzymatic Techniques for the Study of Pathways of Carbohydrate Utilization in the Rumen

Enzymatic techniques were used to study the metabolism of carbohydrates by ruminal bacteria. A direct relationship was observed between the proportions of acetate and propionate formed and the specific activities of the enzymes which participate in forming these acids. An inverse relationship between butyrate formation and butyrate-forming enzymes was observed. The relative activities of succinic dehydrogenase to fumaric reductase, nicotinamide adenine dinucleotide-linked glutamic dehydrogenase to nicotinamide adenine dinucleotide phosphate-linked glutamic dehydrogenase, and pyridine nucleotide-nonlinked lactic dehydrogenase to pyridine nucleotide-linked lactic dehydrogenase were affected by the level of concentrates in the diet. Lactyl coenzyme A dehydrase activity was below the limits of the assay technique in many samples from the alfalfa hay diet, and increased to relatively high levels when concentrates were fed. It is suggested that the enzymatic method will prove valuable for studying the contributions of individual microorganisms to the overall ruminal metabolism, and, with certain limitations, useful for estimating the relative contributions of alternate pathways.     

Mechanism for Regulating the Distribution of Glucose Carbon Between the Embden-Meyerhof and Hexose-Monophosphate Pathways in Streptococcus faecalis

Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.     

Enzymes of Intermediary Carbohydrate Metabolism in the Obligate Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus

Levels of enzymes operative in the Embden-Meyerhof-Parnas (glycolytic) pathway, pentose phosphate cycle, citric acid cycle, and certain other phases of intermediary carbohydrate metabolism have been compared in Thiobacillus thioparus and T. neapolitanus. All enzymes of the glycolytic pathway except phosphofructokinase were demonstrated in both organisms. There were some striking quantitative differences between the two organisms with respect to the activities of the individual enzymes of the glycolytic pathway and the citric acid cycle. Qualitative differences were also found: the isocitrate dehydrogenase activity of T. thioparus is strictly nicotinamide adenine dinucleotide phosphate (NADP)-dependent, whereas that of T. neapolitanus is primarily nicotinamide adenine dinucleotide-dependent, activity with NADP being low; the glucose-6-phosphate dehydrogenase of T. thioparus is particulate, whereas that of T. neapolitanus is partly soluble and partly particulate; the 6-phosphogluconate dehydrogenase of T. thioparus is soluble, that of T. neapolitanus is partly soluble and partly particulate. All enzymes which function in the carbon reduction cycle were present at very high levels. In contrast, enzymes which operate exclusively in cycles other than the carbon reduction cycle were present at low levels. Of the enzymes not operative in the carbon reduction cycle that were examined, isocitric dehydrogenase had the highest specific activity. Both organisms possessed reduced nicotinamide adenine dinucleotide dehydrogenase activity. The qualitative and quantitative aspects of the data are discussed in relation to possible biochemical explanations of obligate autotrophy.     

Changes in glutamate dehydrogenase activity of Chlamydomonas reinhardtii under different trophic and stress conditions

Abstract. Under stress conditions (darkness, nitrogen starvation, high ammonium concentrations, glutamine synthetase and glutamate synthase inhibition) glutamate dehydrogenase animating activity levels of Chlamydomonas cells varied inversely to those of glutamine synthetase. Nitrogen and carbon sources also influenced glutamate dehydrogenase levels in Chlamydomonas , the highest values being found in cells cultured mixotrophically with ammonium, under which conditions glutamate dehydrogenase and glutamine synthetase levels were likewise inversely related. These facts, together with the analysis of internal fluctuations of ammonium, 2-oxoglutarate, and the amino acid pool as well as the variations of certain enzymes involved in carbon metabolism indicate that glutamate dehydrogenase animating activity is adaptative, being involved in the maintenance of intracellular levels of L-glutamate when they cannot be maintained by the GS-GOGAT cycle, and probably more connected with carbon than nitrogen metabolism.     

Oxidative and phosphorylative activity of mitochondria from pea cotyledons during maturation of the seed

Summary The respiration rate and the activity of some mitochondrial enzymes from pea cotyledons have been followed during the final phases of seed development, when the relative water content of the cotyledons dropped from 65 to 13%. Succinate, malate and -ketoglutarate oxidase activity, and succinate and malate dehydrogenase activity per cotyledon increased when the relative water content dropped from 65 to about 55%. A further drop of the relative water content was accompanied by a strong decrease of the activity of the succinate and malate oxidase system, but only a slight decrease of succinate and malate dehydrogenase activity. Mitochondrial fractions from air-dry, mature cotyledons showed a low activity of the succinate and malate oxidase system but their dehydrogenase activity was relatively high. The phosphorylation efficiency and respiratory control gradually decreased during maturation. These results indicate that during maturation of the pea seed certain mitochondrial enzymes partly lose their activity.     

Post-mortem changes in human central nervous tissue and the effects on quantitation of nucleic acids and enzymes.

A study of post-mortem changes in human central nervous tissue has shown that within 100 h of death, no significant change occurs in the amount of nerve cell DNA and nucleolar RNA nor in some membrane-associated enzymes such as succinate dehydrogenase, NADH and NADPH diaphorase, and cytochrome oxidase. Low molecular weight RNA species, probably transfer and messenger RNA are quickly lost, but there is little alteration in ribosomal RNA content. Cytoplasmic enzymes show variable changes; phosphofructokinase activity is rapidly decreased; hexokinase is unaltered but lactate dehydrogenase, pyruvate kinase and glucose-6-phosphate dehydrogenase initially show increases in activity which subsequently decline. Oxygen uptake diminishes quickly. These findings indicate that mechanical alterations in cell structure, following death, render organelles physiologically ineffective long before any significant changes in certain constituent biochemicals are detected. This report emphasizes the great importance necessary in the selection of appropriately time matched post-mortem tissues if accurate comparative studies of many of the cells constituents are to be made.     

Effect of borate on the catalytic activities of muscle glyceraldehyde-3-phosphate dehydrogenase.

The effect of borate on glyceraldehyde-3-phosphate dehydrogenase from human, pig and rabbit muscle was studied. At lower concentration of borate only the dehydrogenase activity is inhibited, reversibly and competitively against NAD. At concentration of borate above 6 mM the plots of 1/v versus borate concentration become nonlinear and the inhibition is extended to the esterase and acetylphosphatase activities. In certain conditions a time-dependent inactivation and reactivation was observed. The direct interaction between borate (if present at concentration of at least 6 mM) and glyceraldehyde-3-phosphate dehydrogenase is postulated, the possible site of the reaction being the histidine residue(s). The esterase activity of the human muscle enzyme and the effect of borate on it are different from the other mammalian enzymes.     

Regulation of phosphoglycerate dehydrogenase levels and effect on serine synthesis in Escherichia coli K-12.

The level of phosphoglycerate dehydrogenase, the first enzyme in the biosynthetic pathway to serine and glycine, has been studied in Escherichia coli grown under different conditions. The enzyme level was not reduced by growth in a medium which contained the end products of the pathway, nor was it elevated when the growth rates was limited by the supply of serine. Elevation of phosphoglycerate dehydrogenase did not occur when charging of tRNA ser was inhibited by serine hydroxamate. However, phosphoglycerate dehydrogenase levels were subject to regulation. Elevated levels of enzyme activity were observed in merodiploids containing multiple copies of the serA gene, and lowered enzyme levels were found in cells grown on carbon sources other than glucose or when certain amino acids were present in the growth medium. The combined effect of these nutritional changes (carbon source and amino acids) reduced the level of phosphoglycerate dehydrogenase to 10 to 12% of that found in wild-type cells and to about 5% of the level in the merodiploids. By using antibody prepared against purified phosphoglycerate dehydrogenase we established that the decrease in enzyme activity reflected decreased amounts of enzyme protein. Constant intracellular concentrations of 3-phosphoglycerate and serine were found in cells with marked differences in phosphoglycerate dehydrogenase activity, indicating that end product inhibition of phosphoglycerate dehydrogenase activity, rather than the amount of the biosynthetic enzymes, is the major factor in regulating the intracellular concentration of serine.     

Kinetic properties of enzymes in particular of yeast alcohol dehydrogenase following their adsorption on polyaminomethylstyrene]

The adsorption of 8 enzymes to polyaminomethylstyrene was studied. While lactate dehydrogenase, alkaline phosphatase and glucose-6-phosphate dehydrogenase exhibit a relatively low affinity to the carrier, alcohol dehydrogenase, glutamate dehydrogenase and urease were found to form stabile complexes with the polymer that are enzymatically active. Adsorbed urease and beta-hydroxybutyrate dehydrogenase, are still active after several weeks; the other preparations lose their activity soon. It can be shown by the example of yeast alcohol dehydrogenase that the activity loss following adsorption is caused possibly by a process of reorientation of already bound enzyme molecules or by the increasing enzyme coverage of the carrier, with the active centres becoming more and more inaccessible for the substrate. During the substrate conversion catalysed by the alcohol dehydrogenase-polyaminomethylstyrene complex, a small amount of the enzyme is again detached from the carrier. The activity rises to a certain extent in the supernatant but drops to zero again. The stability of the adsorbed urease is distinctly increased compared with the dissolved enzyme. For the pH optimum and the KM value there are no differences between the two preparations. Continuous application of polyaminomethylstyrene-bound beta-hydroxybutyrate dehydrogenase and urease, respectively, in a column shows that both preparations have unchanged enzymatic activities even after running times of 5 and 24 days, respectively.     

Short- and long-term enzymatic regulation secondary to metabolic insult in the rat retina

Changes in oxygen and/or glucose availability may result in altered levels of ATP production and amino acid levels, and alteration in lactic acid production. However, under certain metabolic insults, the retina demonstrates considerable resilience and maintains ATP production, and/or retinal function. We wanted to investigate whether this resilience would be reflected in alterations in the activity of key enzymes of retinal metabolism, or enzymes associated with amino acid production that may supply their carbon skeleton for energy production. Enzymatic assays were conducted to determine the activity of key retinal metabolic enzymes total ATPase and Na(+)/K(+)-ATPase, aspartate aminotransferase and lactate dehydrogenase. In vitro anoxia led to an increase in retinal lactate dehydrogenase activity and to a decrease in retinal aspartate aminotransferase activity, without significant changes in Na(+)/K(+)-ATPase activity. In vivo inhibition of glutamine synthetase resulted in a short-term significant decrease in retinal aspartate aminotransferase activity. An increase in retinal aspartate aminotransferase and lactate dehydrogenase activities was accompanied by altered levels of amino acids in neurons and glia after partial inhibition of glial metabolism, implying that short- and long-term up- and down-regulation of key metabolic enzymes occurs to supply carbon skeletons for retinal metabolism. ATPase activity does not appear to fluctuate under the metabolic stresses employed in our experimental procedures.     

Effect of gossypol on few testicular enzymes in mature rats

Effect of oral administration of gossypol acetic acid (15 mg/kg/day) for 10 weeks, on certain enzymes, which may be taken as markers for the different stages of spermatogenesis, was studied in male albino rats. Gossypol produced a significant decrease in hyaluronidase and sorbitol dehydrogenase, while no change was observed in beta-glucuronidase and acid phosphatase. A significant increase in the total lactate dehydrogenase activity was observed in the testis. The possible significance of these findings is discussed.     

Age and the succinate dehydrogenase activity of human cardiac neurons]

Under study was the SDG activity in human heart neurons in postnatal ontogensis. Succinic dehydrogenase was revealed with nitrablue tetrazole after Nachlas, quantitative measurements in preparations were made in a cytospectrophotometer. A regular developmental increase in the SDG activity in human heart neurons was established to have certain fluctuations in different age periods. The most intensive growth of the enzyme activity was found in early childhood, youth and senile age which is related to physiological state of the organism and the cardio-vascular activity.     

Production of quinoprotein d-glucose dehydrogenase in the culture medium of Acinetobacter calcoaceticus

On addition of low concentrations (0.005%) of Triton X-100 to a mineral medium supplemented with 0.5% heptadecane, a marked stimulation of growth rate was observed for Acinetobacter calcoaceticus strains able to grow on alkanes while appreciable amounts of soluble quinoprotein d-glucose dehydrogenase [d-glucose: (pyrroloquinoline-quinone) 1-oxidoreductase, EC 1.1.99.17] were found in the culture medium. At higher Triton X-100 concentrations (0.04%), still larger amounts of d-glucose dehydrogenase and also cytoplasmic enzyme activities appeared in the culture medium. Although combinations of other carbon sources plus non-ionic detergents also produced these enzymes in the medium, the combination of heptadecane and Triton X-100 gave higher levels and had a stabilizing effect on d-glucose dehydrogenase. Therefore, by using this combination and culturing within certain pH limits, a stable enzyme solution, having already a high specific activity, is produced while the cell harvesting and disruption steps can be circumvented. The results indicate that d-glucose dehydrogenase in this organism is a periplasmic enzyme, coupled to a cytochrome b.