Steroidogenesis despite a variant metabolism in primary cultures of pig granulosa cells

Enzymic changes in primary cultures of granulosa cells over 9 days were measured and compared with changes occurring during follicular development in vivo. Characteristic of in vivo development of granulosa cells was a large increase in activities of the NADP+-dependent isocitrate, glucose-6-phosphate dehydrogenases and malic enzyme, and smaller increases in the activities of the NAD+-dependent lactate and malate dehydrogenases. In vitro, the NAD+-dependent dehydrogenases increased in activity, while the NADP+-dependent enzymes showed transient or no changes. Despite the uncharacteristic metabolism, granulosa cells in culture could synthesize steroids. Our results suggest that the cells in vitro and in vivo use different metabolic pathways to support syntheses dependent on reducing equivalents.     

Changes in the activity of selected enzymes during starvation of Physarum polycephalum

The levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and cyclic phosphodiesterase activities were examined in growing and starving plasmodia of Physarum polycephalum. The activities of lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase decreased whereas that of cyclic phosphodiesterase increased. The change in activity of lactate dehydrogenase was the result of the variation of the activity of a single enzyme quite similar to the lactate dehydrogenases of higher animals.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

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)     

Liver dehydrogenase levels in rainbow trout, Salmo gairdneri, fed cyclopropenoid fatty acids and aflatoxin B1

Cyclopropenoid fatty acids in the diet of rainbow trout caused significant reductions in liver protein and activity of glucose-6-phosphate dehydrogenase, NADP-linked isocitrate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. Changes in total activity were usually accompanied by similar changes in specific activity. The activity of glucose-6-phosphate dehydrogenase appeared to be more sensitive to the ingestion of cyclopropenoid fatty acids than the other dehydrogenases studied. Feeding 20 ppb aflatoxin B(1) to rainbow trout did not significantly change the activity of the dehydrogenases except for a small increase in the activity of glucose-6-phosphate dehydrogenase after 21 days of feeding. Relationships of these changes to the cocarcinogenicity of cyclopropenoid fatty acids and the carcinogenicity of aflatoxin are discussed.     

Ziram, a sulfhydryl reagent and specific inhibitor of yeast mitochondrial dehydrogenases.

The fungicide zinc dimethyldithiocarbamate (ziram) is a sulfhydryl reagent which inhibits specifically the growth of the yeast Saccharomyces cerevisiae on nonfermentable substrates. In isolated mitochondria, the uncoupled as well as the state 3 oxidations of succinate, α-ketoglutarate, ethanol, and malate plus pyruvate are sensitive to ziram concentrations of 10 to 30 μm. The oxidations of isocitrate, of external NADH, of α-glycerophosphate, and of ascorbate plus tetramethylphenylenediamine exhibit a lower sensitivity to ziram. Succinate, α-ketoglutarate, and pyruvate dehydrogenases activities are 50% inhibited by concentration of ziram lower than 10 μm. At the same concentrations, neither the mitochondrial transports of succinate, ADP, or phosphate nor oxidative phosphorylation and adenosine triphosphatase activities are modified. The kinetic study of the inhibition by ziram of succinate dehydrogenase activity shows that ziram is noncompetitive with succinate and produces sigmoidal inhibitions of state 3 and of uncoupled oxidation of succinate by intact mitochondria. Inhibition of succinate:phenazine methosulfate oxidoreductase activity yields exponential kinetics. However sigmoidal-type inhibition is observed when succinate dehydrogenase activity is stimulated by ATP.     

Enzymes and Nucleotides in Virions of Rous Sarcoma Virus

In addition to the previously described deoxyribonucleic acid (DNA) polymerase, DNA ligase, DNA exonuclease, and DNA endonuclease activities, purified virions of Schmidt-Ruppin strain of Rous sarcoma virus (SRV) have nucleotides and nucleotide kinase, phosphatase, hexokinase, and lactate dehydrogenase activities. The SRV virions have no glucose-6-phosphate dehydrogenase activity. All enzyme activities, but glucose-6-phosphate dehydrogenase and adenosine triphosphatase, were increased by disruption of the virions. The DNA polymerase, DNA ligase, and hexokinase activities had a higher specific activity in purified virion cores. It is suggested that during assembly virions of SRV may pick up cytoplasmic components which bind to virion proteins. The role of these components in viral replication is not known at present.     

Synthesis of immunoglobulin conjugates with dehydrogenases

Three methods of synthesis of immunoglobulin conjugates with malate, lactate and glucose-6-phosphate dehydrogenases, involving the sodium metaperiodate oxidation of immunoglobulin carbohydrate component, use of water-soluble carbodiimide and the one-step glutaraldehyde technique, were compared. The glutaraldehyde method was shown to give immunoglobulin-dehydrogenase conjugates with high catalytic and immunochemical activity, which may be useful for enzyme-immunoassay.     

Brain enzyme histochemistry following stabilization by microwave irradiation

Summary The activities of various enzymes present in brain homogenates were assayed biochemically (a) with no pretreatment, (b) following a standard microwave treatment in saline and (c) after a standard microwave treatment in formalin. All enzyme activity was lost after the microwave — formalin in treatment. Following microwave — saline treatment, the activities of alkaline phosphatase, 5-nucleotidase, isocitrate and succinate dehydrogenases were reduced. In contrast, the activities of lactate and malate dehydrogenases were unchanged, and that of acetylcholinesterase apparently increased.Analogous outcomes were seen following attempted histochemical demonstrations of these enzymes. Thus satisfactory histochemical demonstration of all enzymes was achieved (except with alkaline phosphatase, lactate and malate dehydrogenases) following the microwave-saline pretreatment. Since acid phosphatase, catalase and peroxidase were also successfully demonstrated, it seems that microwave-saline pretreatments permit both retention of sufficient enzyme activity for histochemical demonstration to occur and retention of sufficient structural integrity for critical morphological investigations. Since the failure to stain the sites of lactate and malate dehydrogenases is not due to microwave inactivation of these enzymes, their demonstration may be possible by varying the staining procedures.     

Electrophoretic studies of several dehydrogenases in relation to the cold tolerance of alfalfa.

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening conditions to determine the solubility characteristics and relationships of several dehydrogenases to cold tolerance.Soluble enzymatic proteins, extracted with three extractants, from lyophilized crown and root tissues, were separated by polyacrylamide disc gel electrophoresis.Gels assayed for glutamate, NAD-malate, NADP-malate, isocitrate, lactate, 6-phosphogluconate, and glucose-6-phosphate dehydrogenases showed quantitative differences in isoenzymes that were influenced by cultivar, extractant, and environmental differences.For both cultivars, enzyme activity was lowest during summer, increased in winter, and decreased during dehardening. Dehydrogenase activity, therefore, was closely associated with the fluctuations in soluble protein concentration, which were related to environmental changes and cold tolerance.Additional isoenzymes of isocitrate, lactate, and glucose-6-phosphate dehydrogenases were detected in the winter samples of both cultivars; however, most of the qualitative differences observed were generally due to the differential solubilities of isoenzymes in the three extractants.Comparison of data obtained from the use of frozen and unfrozen extracts indicated differential stabilities of the dehydrogenases to freezing in the different extractants. Glutamate, NAD-malate, and NADP-malate dehydrogenases were fairly stable to freezing whereas isocitrate, lactate, 6-phosphogluconate, and glucose-6-phosphate dehydrogenases were labile. Detectable levels of the latter dehydrogenases in frozen extracts were evident only in certain extracts of winter samples, indicating the importance of the nature of the extraction medium in protecting against enzyme denaturation.Since both cultivars showed similar changes in dehydrogenase activities at most times, the increased enzyme levels during winter coincided with increased levels of soluble protein and soluble sugars, which are indicative of the broad spectrum of metabolic changes involved in the attainment of the cold-tolerant state.     

Enzyme histochemistry of the sheep uterus during the oestrous cycle

Enzyme histochemical techniques were applied to frozen sheep uteri from different stages of the oestrous cycle. The localization and activities of succinate, lactate, glucose-6-phosphate, and isocitrate (NADP+) dehydrogenases and acid and alkaline phosphatases were studied in the luminal and glandular epithelia, caruncle and myometrium. Enzyme activity in the sections was scored on a scale of 0--5. In general the enzyme activity in the uterine caruncles and epithelia was higher than in the myometrium. The myometrium did not show any alkaline phosphatase activity and isocitrate dehydrogenase (NADP+) activity was negligible. The low activities of acid phosphatase and lactate dehydrogenase and the moderate levels of glucose-6-phosphate and succinate dehydrogenases in the myometrium were constant. The caruncular tissue showed high levels of phosphatases and glucose-6-phosphate dehydrogenase, moderate levels of lactate and succinate dehydrogenases, and low levels of isocitrate dehydrogenase (NADP+) throughout the oestrous cycle. Much lower phosphatase and isocitrate dehydrogenase (NADP+) levels were found in the epithelium of deep glands compared with superficial glands. The high activity of acid and alkaline phosphatases in the luminal epithelium and the superficial glands was constant from mid-cycle to ovulation, but a significant decrease was observed immediately after ovulation. The level of dehydrogenases in epithelia was generally high and did not change during the oestrous cycle.     

The quantitative histochemistry of a chemically induced ependymoblastoma. I. Enzymes

Enzymes from several metabolic pathways were studied quantitatively in homogenates and in homogeneous areas of frozen-dried cryostat sections of an experimental, mouse ependymoblastoma, mouse mammary carcinoma, and mouse melanoma, growing as transplants in mouse brain. Micro analyses were performed in fiveto sixfold replicates on portions of tumour with a dry weight of 0·03-0·2 μg. A close resemblance of the enzyme spectrum of the ependymoblastoma to that of immature brain was noted. Hexokinase and malate dehydrogenase were lower and lactate, glucose-6-phosphate, and NADP⁺-linked isocitrate dehydrogenases, and β-glucuronidase higher in the ependymoblastoma than in whole, adult mouse brain. Mouse mammary carcinoma had a higher level of hexokinase and lower levels of lactate and glucose-6-phosphate dehydrogenases and β-glucuronidase than ependymoblastoma. The concentration of malate dehydrogenase was lower and that of lactate, glucosed-6-phosphate, and NADP⁺-linked isocitric dehydrogenases was higher in the melanoma than in the ependymoblastoma. β-Glucuronidase levels were similar in these two neoplasms. It is suggested that the relatively high levels of several NADP⁺-linked enzymes in the ependymoblastoma may be related to increased capacity for lipid synthesis.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

LEVELS OF CEREBRAL CORTICAL GLYCOLYTIC AND CITRIC ACID CYCLE METABOLITES DURING HYPOGLYCEMIC STUPOR AND ITS REVERSAL

Abstract— Blood glucose, cerebral cortical glucose, and eight metabolites of the glycolytic pathway and citric acid cycle were measured during insulin hypoglycemic stupor and during the first 100s after glucose administration. In hypoglycemic mice that had lost righting ability, blood and brain glucose were decreased 89% and 96% respectively, but glucose-6-phosphate fell only 23%. Other glycolytic and citric acid cycle intermediates were decreased 31–77%. Fructose bisphosphate, 3-phosphoglycerate and phosphopyruvate fell more than glucose-6-phosphate, but less than pyruvate and lactate. Citrate fell less than a-ketoglutarate and malate. These results suggest that in severe hypoglycemia there is a decrease in brain glucose utilization, mediated by phosphofructokinase, but probably caused by decreased neuronal activity. An intravenous injection of glucose restored brain glucose to 75% of normal within 10s and caused return of righting ability within 60s. Glucose-6-phosphate, fructose bisphosphate, 3-phosphoglycerate, and phosphopyruvate rose to normal or near normal levels within 60s, whereas pyruvate, lactate, citrate, ã-ketoglutarate, and malate changed little in this period. This suggests that although glucose given to hypoglycemic animals rapidly enters the glycolytic pathway in brain (and behavior is almost normal), total neuronal activity, and hence overall glucose metabolism, remains subnormal for several minutes.     

Oxidative enzymes in the sebaceous glands of the domestic cat

Synopsis The distribution and activities of several oxidative enzymes in various regions of the sebaceous glands of the domestic cat have been studied. The results obtained emphasize the outstanding importance of NADP-linked dehydrogenases for lipogenesis during sebum production. In particular, the reactions for glucose-6-phosphate dehydrogenase were very strong. Among the NAD-linked dehydrogenases investigated, lactate dehydrogenase showed strong activity in the peripheral cells of the sebaceous gland. The reactions for cytochrome oxidase and succinate dehydrogenase were weaker.     

Catalytic activity and thermostability of dehydrogenase conjugates with cortisol and progesterone.

The conjugates of glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase with progesterone and cortisol, containing 1-40 steroid molecules per enzyme molecule, were obtained by the reactions of N-succinimide esters of the 3-[O-(carboxymethyl)oximes)] of cortisol and progesterone with a protein in a water-DMFA (10%) medium. The catalytic activity and thermostability of dehydrogenases and their steroid conjugates were kinetically studied. The effects of the modification degree on the activity and thermostability of dehydrogenases by their hydrophobization were studied and discussed. Practical recommendations for using the dehydrogenase-steroid conjugates in enzyme immunoassay are given.     

The activity of some intracellular oxidative enzymes in the thyroid follicular epithelium of Xenopus laevis Daud. in experimental long-term hypokinesia.

Histoenzymological methods were applied to examine the activities of previously little studied intracellular oxidative enzymes in the cells of the thyroid follicular epithelium of Xenopus laevis Daud. specimens kept in aquarium. Succinate, lactate, alpha-glycerophosphate, glucose-6-phosphate and reduced NAD and NADP dehydrogenases and cytochrome oxidase were studied. In comparison with other chordate species a very strong activity was revealed particularly by lactate dehydrogenase. The data obtained suggest that the metabolism in thyroid cells of the motionless Xenopus is based on glycolysis mainly.     

On the regional histology and histochemistry of the epididymis of the camel (Camelus dromedarius).

Distinct morphological regions, initial, middle and terminal segments, were distinguishable histologically; the middle segment was further subdivided into proximal, intermediated and distal parts. PAS-positive, diastase-resistant reaction was detected in the blood vessels, subepithelial tissue and stereocilia of all segments. Acid phosphatase was demonstrated in the epithelial cells with the highest activity being in the proximal part of the middle segment. Non-specific esterase gave a similar reaction but the strongest activity was in the terminal segment. Alkaline phosphatase, adenosine triphosphatase and adenosine monophosphatase were of similar activity in the subepithelial tissue, blood vessels, stereocilia and luminal contents; the strongest reaction occurred in the middle segment. Lactate, succinate, glutamate and glucose-6-phosphate dehydrogenases were examined; LDH was more active than the others particularly in the terminal segment. Some reaction was found in the epithelial cells, subepithelial tissue and luminal contents.     

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.     

Enzyme histochemical studies on rat lungs 2. Normal enzyme distribution in the alveolar tissue of the mature lung

Synopsis The activity and distribution of the following eighteen oxidative and hydrolytic enzyme systems have been investigated in the lung of the adult rat: reduced NAD dehydrogenase, reduced NADP dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, adenosine triphosphatase, 5-nucleotidase, non-specific esterase, cytochrome oxidase and -glucuronidase.The low concentration of cells in sections of inflated lung may have made histochemical demonstration of some enzymes impossible because the enzyme concentration was below that detectable by the method employed.The carboxylic acid cycle and the hexose monophosphate shunt were potentially active but fatty acid metabolism was not indicated.The granular reaction sometimes encountered in alveolar cell cytoplasm may be useful for differentiating alveolar cell types, but further cytochemical studies are required to resolve the possible metabolic differences of alveolar cells.