Activity and Isoenzyme Pattern of Lactate Dehydrogenase in Neurons and Astroblasts Cultured from Brains of Chick Embryos

Primary cultures of neurons and glial cells (astroblasts) prepared from brains of 8-day-old and 15-day-old chick embryos, respectively, were grown for periods between 3 and 19 days. Specific activity of lactate dehydrogenase (LDH) increased in both types of cultures as a function of time and was always significantly higher in glial cells than in neurons. Glial cell extracts were found to contain predominantly the anaerobic isoenzymatic form of LDH (LDH-H4), and this pattern did not change over a period of 19 days. Cultured neurons contained predominantly the aerobic isoenzymatic form LDH-H4, and there was a progressive appearance of all other isoenzymes over an 8-day period. These results support the hypothesis of a different energy metabolism in neurons and glia.     

Effects of thyrotoxicosis on mitochondrial enzymes of rat soleus.

Cytochrome oxidase, glycerol-3-phosphate dehydrogenase, and succinate dehydrogenase were measured in mitochondrial fractions obtained from rat soleus muscle of control and 8 week T3 + T4 treated animals. Under these conditions of prolonged treatment, there is a five-fold increase in the specific activities of both cytochrome oxidase and glycerole-3-phosphate dehydrogenase. Significant increases in total cellular mitochondrial content and enzyme activities were observed in T3 + T4 treated animals as compared to controls. These results indicate that thyrotoxicosis can induce selective changes in mitochondrial enzymes in slow twitch red (Type I) muscle fibers.     

Changes in respiratory activities during the cell-cycle of the fission yeast Schizosaccharomyces pombe 972h? growing in the presence of glycerol

1. The specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase, and NADPH-cytochrome c oxidoreductase in mid-exponential-phase batch cultures of glycerol-grown Schizosaccharomyces pombe indicated that the organisms were catabolite-de-repressed. 2. In cultures growing synchronously in the presence of glycerol as sole carbon source, the respiration rate showed two abrupt increases at about 0.45 and 0.95 of the cell-cycle and remained constant in the periods between successive rises. 3. Catalase, succinate dehydrogenase, NADH-cytochrome c oxidoreductase and acid p-nitrophenyl-phosphatase all showed peak patterns of expression in synchronous cultures. 4. Cytochrome c oxidase and cytochromes a+a(3) both showed step patterns of expression with two rises per cell-cycle. 5. Cytochromes c(548), b(554) and b(560) all followed similar time-courses in step patterns of expression, but these were distinct from, and more complex than, that of cytochromes a+a(3). 6. These results are compared with those previously obtained with glucose-grown cultures, and the part played by catabolite repression in the expression of respiratory activities in the cell-cycle is assessed.     

Histochemical study on the distribution of some enzyme activities in the vagal and facial lobes of the goldfish, Carassius auratus.

The histochemical localization of six enzymic activities (acetylcholinesterase, pseudocholinesterase, monoamine oxidase, lactate dehydrogenase, succinate dehydrogenase and glucose-6-phosphate dehydrogenase) has been studied in the vagal and facial lobes of the goldfish, Carassius auratus. These encephalic centers are hypertrophic in Cyprinidae, corresponding to the dominance of gustatory function. Acetylcholinesterase shows a complex laminar distribution in the vagal lobes and a peculiar cellular localization in vagal motor neurons. Monoamine oxidase activity is mainly evident in fibrous tracts coming to or leaving from the lobes. Among oxidative enzymes examined, lactate dehydrogenase and succinate dehydrogenase exhibit distribution patterns respectively similar to those observed for acetylcholinesterase and monoamine oxidase. Some features on enzymes distribution in the gustatory centers of Carassius are in agreement with the enzymatic patterns well known in higher vertebrates.     

Myoglobin synthesis in cell cultures of red and white muscle

Myoglobin synthesis was compared in cell cultures of leg (red) and breast (white) muscle of chick embryos. In leg muscle cultures a rapidly increasing amino acid incorporation into myoglobin begins two days after muscle cell fusion; in breast muscle cultures no comparable increase was observed. This qualitative difference in cultures of the two muscle cell types provides possibilities for the further study of the mechanism of myoglobin synthesis.     

Expression of differentiated activities in reaggregated brain cell cultures.

Dissociated fetal mouse brain cells are allowed to reassociate in rotation culture to form aggregates. After several weeks these reaggregated brain cell cultures show markedly increased specific activities of monoamine oxidase, lactate dehydrogenase, and the brain-specific protein S-100, while catechol-O-methyltransferase activity increases slightly. Similar changes in these activities are found during mouse brain maturation. The amounts of monoamine oxidase, catechol-O-methyltransferase, and S-100 were also determined in surface cultures of fetal mouse brain cells, as well as glioma and neuroblastoma cell lines. The fetal brain and glial cell cultures possess much higher activities than the cultured neuroblastoma cells. However, lactate dehydrogenase activity was highest in the glioma and lowest in the surface cultures of fetal brain cells.     

Histochemical studies of fibroblasts from patients with Menkes kinky hair disease and Wilson's disease

Summary Cultured fibroblasts from Menkes kinky hair disease patients showed markedly reduced succinate dehydrogenase and amine oxidase activities. Cytochrome oxidase activity, however, was greatly reduced in some cells and almost normal in others. Cultured fibroblasts from patients with Wilson's disease showed moderately reduced succinate dehydrogenase and cytochrome oxidase activities. Amine oxidase activity was only slightly reduced when compared to that of normal. These results indicated that the histochemical phenotype observed in fibroblasts from patients with Menkes kinky hair disease and Wilson's disease were distinctly different from each other and from normal fibroblasts.     

Mitochondrial respiratory and antioxidative enzyme activities in broiler meat in relation to age and gender of the animals

Colour is an important quality parameter of broiler meat influencing the consumer buying behaviour. The alterations of the colour after slaughter are related to the oxidative status of the tissue. This in turn is influenced by an interaction between the mitochondria and the antioxidative enzymes. In this study, breast muscles were collected from hens and cocks of a commercial line slaughtered at the ages of 28 and 41 day. Analysis of the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) was performed with samples obtained 20 min and 48 h after slaughter (post mortem, p.m.), whereas the mitochondrial respiratory activity was analysed in permeabilised breast muscle fibres collected 20 min p.m. The carcass characteristics of breast muscle and leg weight as well as breast yield were significantly higher, and the leg yields lower, in the 41-day-old broiler. The 28-day-old hens and cocks had comparable carcass characteristics (P > 0.05), whereas 41-day-old cocks had significantly higher carcass, breast and leg weight in comparison to the hens. The pH20 min p.m. and the L*48 h p.m. were significantly higher, and the a* and b* values of the 20 min and 48 h p.m. samples as well as the drip loss were significantly lower in the 41-day-old broiler. Mitochondrial respiratory rates were comparable (P > 0.05) between the 28- and 41-day-old cocks and hens. The same result could be found with regard to the activities of the SOD, GPx and GR except for lower activities of the SOD20 min p.m. and higher of the GR48 h min p.m. in the 41-day-old broiler. The concentrations of thiobarbituric acid-reactive substances were generally higher in the breast muscles of the 41-day-old broiler. Assorting the data according to their mean pH20 min p.m. indicates a positive influence of higher pH values (>6.34) on the mitochondrial function, whereas a low pH20 min p.m. results in tendentially and significantly higher activities of the antioxidative enzymes and drip loss values. These results indicate a relation between the meat quality and the oxidative metabolism as well as antioxidative capacity of the meat.     

Development of glial cells in primary cultures: Energy metabolism and lactate dehydrogenase isoenzymes

Primary cultures of glial cells prepared from brains of newborn rats were grown for periods of 1–5 weeks. After a proliferative phase of between 2 and 3 weeks, the cultures were maintained in stationary phase, during which a significant increase of oxygen consumption and of the activities of lactate dehydrogenase, succinate dehydrogenase, and mitochondrial glycerolphosphate dehydrogenase could be observed. Furthermore, qualitative changes in the lactate dehydrogenase isoenzyme pattern were found with time, characterized by a shift toward an enhanced synthesis of H subunits. A similar development was found in comparing the LDH isoenzyme pattern in the brain of 15-day-old rat embryo with those of newborn and adult rat brains. It is suggested that some aspects of maturation of glial cells in culture are comparable to those occurring in whole brain in vivo, namely a shift towards an enhanced aerobic metabolism.     

Succinic Dehydrogenase and Cytochrome Oxidase Activities in Cell Cultures

Succinic dehydrogenase and cytochrome oxidase have been assayed in permanent cell lines (HEP 1, HEP 2, and HLM), in short-term cultures of chick embryo heart cells, and in various tissues. Their activities in different cells are compared by relating them to deoxyribonucleic acid. They are very low in HEP 1, HEP 2, and HLM cells by comparison with the activities in any normal tissues examined. All the succinic dehydrogenase was shown to be located in the mitochondria of the permanent cell lines by staining with tetrazolium derivatives. Both enzymes were more active in tissues of 19-day chick embryos than in those of 11- or 14-day embryos. The increasing activities found during normal development were quickly curtailed or reversed when heart cells were grown as monolayer cultures. Digitonin-treated mitochondria produced preparations with much higher activities of cytochrome oxidase than untreated samples. Activities measured in this way were again very much lower in HEP 1, HEP 2, and HLM cells than in the normal tissues. From the derived ratio of cytochrome oxidase:succinic dehydrogenase, it was apparent that cytochrome oxidase is diminished to a greater extent than succinic dehydrogenase in both permanent cell lines and short-term cultures, by comparison with the corresponding activities in embryonic and adult tissues. The features common to the metabolism of proliferating cells in vitro and malignant cells are discussed.     

Comparative enzyme histochemical study on the visceral yolk sac endoderm in the rat in vivo and in vitro

Histochemical study of the visceral yolk-sac endoderm of the rat was performed in vitro (whole-embryo culture for 24, 48 and 72 h explanted at 9.5 days of gestation) and in vivo (10.5, 11.5 and 12.5 days of gestation) in order to compare the distribution and activity of various enzymes involved in the digestion and energy metabolism in both systems. It was shown that, both in vitro and in vivo gamma-glytamyltransferase and dipeptidylpeptidase IV are demonstrable in the apical cell membranes (membrane-bound hydrolases), while acid phosphatase, dipeptidylpeptidases I, II and acid beta-galactosidase are concentrated in the supranuclear vacuoles (lysosomal hydrolases), and cytoplasmic lactate dehydrogenase and mitochondrial enzymes (succinate dehydrogenase, NAD-dependent isocitrate dehydrogenase, cytochrom oxidase) are localized in the whole cytoplasm and mainly in the apical cytoplasm, respectively, of the visceral yolk-sac epithelium. In vivo, the activity of all enzymes increased until 12.5 days, but in vitro, this activity increased only until 48 h after the start of culture (corresponding to 11.5 days in vivo). Comparison of the yolk sacs at 10.5 and 11.5 days in vivo with those after 24 and 48 h in vitro showed that the activities of all the investigated enzymes were almost identical. Yolk sacs which were cultured for 72 h showed lower activities of lysosomal and mitochondrial enzymes than those at 12.5 days in vivo. It is concluded that the digestive function and energy metabolism of the visceral yolk-sac epithelium are almost identical in vitro and in vivo at 10.5 and 11.5 days.     

Carbohydrate metabolism of goldfish (Carassius auratus L.)

Summary The effect of hypoxia was studied in cold (15°C) and warm (30°C) acclimated goldfish. The hypoxic thresholds, defined as the lowest sustainablePO₂ were found to be 1.6 and 4.0 kPa O₂ at, respectively, 15°C and 30°C. At these levels the fish did not loose either weight or appetite over a 2-months period. While during starvation under normonic conditions a significant weight loss and breakdown of lactate dehydrogenase (90%) was observed, no such changes were found in fed hypoxic animals. In red lateral muscle, white epaxial muscle and liver of goldfish from 4 differently acclimated groups the maximal activities were measured of: glycogen phosphorylase, hexokinase, malate dehydrogenase, glycerol-3-P dehydrogenase, glucose-6-P dehydrogenase, malic enzyme, succinate oxidase, pyruvate carboxylase, phosphoenol-pyruvate carboxykinase, fructose-bisphosphatase and glucose-6-phosphatase. Thermal compensation, according to Precht's typology, was predominantly observed in red muscle and to a lesser extent in white muscle. The liver glucose-6-P dehydrogenase showed a strong inverse response, which points to enhanced synthetic activity at the higher temperature. Hypoxia acclimation exerted weaker responses at 15°C than at 30°C. Changes in liver enzyme activities suggest depressed protein synthesis and enhanced gluconeogenesis in hypoxic animals. In muscle of 30°C-acclimated goldfish hypoxia induces a significant increase of succinate oxidase activity, indicating adaptation of the aerobic energy metabolism. The occurrence of pyruvate carboxylase, never before observed in vertebrate muscle, probably plays an important role in pyruvate catabolism. Because its action produces oxalo-acetate, the enzyme may stimulate pyruvate oxidation and thus prevent early lactate accumulation. Since all gluconeogenic enzymes were shown to be active in goldfish muscle, the possible occurrence of gluconeogenesis in muscle (albeit at low rate) must be accepted. Enzyme activities in goldfish muscle were compared with literature data for a number of other fish species. This comparison indicates that maximal glycolytic flux in goldfish muscle tissue is rather low, although muscular glycogen levels are very high. It is suggested that this is part of the gold-fish's strategy to cope with hypoxia.     

Methods of microphotometric assay of succinate dehydrogenase and cytochrome c oxidase activities for use on human skeletal muscle

Microphotometric assay media for the measurement of succinate dehydrogenase (SDH) and cytochrome oxidase activities in sections of human skeletal muscle have been developed. The optimal constitution of these media was determined experimentally. Factors investigated include the effects of substrate concentration, pH, use of different electron acceptors and electron donors, influence of intermediate electron carriers and tissue-stabilizing agents, effects of inhibitors, the extent of endogenous and non-specific reactions and the linearity of the reactions during the time course of the assays. Optimal assay media (SDH) contained 130 mM succinate, 1.5 mM Nitro Blue tetrazolium, 0.2 mM phenazine methosulphate and 1.0 mM sodium azide in 0.1 m phosphate buffer, pH7.0. Cytochrome oxidase was optimally assayed in media containing 4 mM diaminobenzidine and 100 microns cytochrome c. Reactions in individual muscle fibers were found to be linear for incubation times up to 10 min in SDH assays and for more than 15 min in cytochrome oxidase determinations. Some potential uses of these microphotometric assays in the investigation of human metabolic muscle disorders are discussed.     

Effect of triamcinolone administration on content of flavins in rabbit liver

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH : lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductace EC 1.6.99.3) and -amino acid oxidase ( -amino acid : oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Effect of triamcinolone administration on content of flavins in rabbit liver.

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductase EC 1.6.99.3) and D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Effect of vagotomy on glycogen content, succinate dehydrogenase, cytochrome-c-oxidase and lactate dehydrogenase activities in myocardium in experimental aortostenosis

The glycogen contents, succinate dehydrogenase, cytochrome-c-oxidase and lactate dehydrogenase activities in tissues of cardiac left cavity of vagotomized and nonvagotomized rabbits were studied two days and two weeks after aortostenosis and false operation. It is established that in vagotimized rabbits the prolongation of the hyperfunction period causes a less pronounced decrease in the glycogen level and a higher level of the succinate dehydrogenase and cytochrome-c-oxidase activities. A conclusion is drawn that heart hyperfunction under conditions of vagotomy is accompanied by a less pronounced inhibition of aerobic and insignificant intensification of anaerobic oxidation processes as compared to their changes in rabbits with the heart maintained innervation.     

Respiratory chain enzymes in muscle of endurance athletes: effect of L-carnitine.

The effects of L-carnitine on respiratory chain enzymes in muscle of long distance runners were studied in 14 athletes. These subjects received placebo or L-carnitine (2 g orally b.i.d.) during a 4-week period of training. Athletes receiving L-carnitine showed a significant increase (p < 0.01) in the activities of rotenone-sensitive NADH cytochrome c reductase, succinate cytochrome c reductase and cytochrome oxidase. In contrast, succinate dehydrogenase and citrate synthase were unchanged. No significant changes were observed after placebo administration. The levels of both total and free carnitine from athletes receiving placebo were significantly decreased (p < 0.01) after treatment. By contrast, total and free carnitine levels were markedly increased (p < 0.01) after supplementation with L-carnitine. Our results suggest that L-carnitine induces an increase of the respiratory chain enzyme activities in muscle, probably by mechanisms involving mitochondrial DNA.     

Synthesis of glycolytic and peroxisomal enzymes in Tetrahymena following a change in culture conditions

The specific activity of a peroxisomal enzyme, lactate oxidase, and of pyruvate kinase and lactate dehydrogenase, which are not peroxisomal, increased rapidly when shaken cultures of Tetrahymena were transferred to conditions of oxygen restriction and supplemented with glucose. Two other peroxisomal enzymes, catalase and TPN-linked isocitrate dehydrogenase, did not increase substantially, nor did succinate dehydrogenase. The increases were reduced if glucose was not added at the time of transfer, and were prevented by actinomycin D or cycloheximide, but not by chloramphenicol. The results suggest an involvement of peroxisomes in the metabolism of glycolytic endproducts when the availability of oxygen to the cell is limiting.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.     

Oscillations of enzyme activities during the cell-cycle of a glucose-repressed fission-yeast Schizosaccharomyces pombe 972h

1. Increased specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase and malate dehydrogenase were observed during glucose de-repression of Schizosaccharomyces pombe. 2. The cell-cycle of this organism was analysed by three different methods: (a) harvesting of cells at intervals from a synchronous culture, (b) separation of cells by rate-zonal centrifugation into different size classes and (c) separation of cells by isopycnic-zonal centrifugation into different density classes. 3. Measurement of enzyme activities during the cell-cycle showed that all the enzymes assayed [cytochrome c oxidase, catalase, acid p-nitrophenylphosphatase, NADH-dehydrogenase, NADH-cytochrome c oxidoreductase, NADPH-cytochrome c oxidoreductase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase (NADP) and fumarate hydratase] show periodic expression as ;peaks'. 4. Cytochrome c oxidase shows a single maximum at 0.67 of a cycle, whereas succinate dehydrogenase exhibits two maxima separated by 0.5 of a cell-cycle. 5. All other enzymes assayed showed two distinct maxima per cell-cycle; for catalase, malate dehydrogenase and NADPH-cytochrome c oxidoreductase there is the possibility of multiple fluctuations. 6. The single maximum of cytochrome c oxidase appears at a similar time in the cycle to one maximum of each of the other enzymes studied, except for NADH dehydrogenase. 7. These results are discussed with reference to previous observations on the expression of enzyme activities during the cell-cycle of yeasts.