Fumarate reductase in the control of heme biosynthesis.

A drug-induced stimulation of heme biosynthesis in mouse liver was accompanied by altered fumarate metabolism. In liver homogenate, fumarate 1,4-C(14) was incorporated, via succinate and succinyl coenzyme A, into heme at an accelerated rate. This pathway of fumarate utilization was inhibited by acetoacetate but not by beta-hydroxybutyrate. Fumarate reduction to succinate required reduced nicotinamide adenine dinucleotide. The enzyme fumarate reductase is suggested as a link between terminal oxidation and cellular control of the heme biosynthetic pathway.

Chemically induced porphyria: prevention by prior treatment with phenobarbital.

The excessive induction of hepatic delta-aminolevulinic acid synthetase in rats after the administration of porphyria-inducing compounds is prevented by prior treatment with phenobarbital. Studies in vivo and in vitro indicate that phenobarbital prevents the induction of chemical porphyria by increasing the rate of detoxification of inducers by way of drug-metabolizing enzymes of the hepatic endoplasmic reticulum.

Carbon dioxide abolishes the reverse Pasteur effect in Leishmania major promastigotes.

The products released by Leishmania major promastigotes incubated with [1-13C]glucose as sole exogenous carbon source were identified using nuclear magnetic resonance (NMR). Under aerobic (95% O2/5% CO2) conditions, acetate, succinate, and small amounts of pyruvate, D-lactate, and glycerol were released in addition to CO2. Under anaerobic (95% N2/5% CO2) conditions, the relative amounts of products formed changed and alanine was also released. The changes in the rates of glucose consumption and product formation during the aerobic to anaerobic transition were measured. Under hypoxic conditions (O2 less than 0.2%), glucose consumption was decreased by about 50%. Under completely anaerobic conditions (100% N2), glucose consumption almost ceased (a total reverse Pasteur effect). The inclusion of 5% CO2 in the gas phase restored hypoxic and anaerobic glucose consumption to the aerobic rate, and increased production of succinate, pyruvate, and D-lactate. Thus, CO2 and very low concentrations of O2 have strong regulatory effects on L. major glucose metabolism. A quantitative carbon balance showed that the NMR-identified products accounted for only about 25% of the glucose carbons consumed under aerobic conditions. CO2, measured as the release of 14CO2 from [U-14C]glucose, accounted for an additional 25% of the glucose consumed. About 11% of the glucose carbon was incorporated into trichloroacetic acid-insoluble products, mostly lipid. Large amounts of label from [U-14C]glucose were incorporated into the intracellular pools of alanine, glutamate, glutamine, and aspartate, indicating that CO2 from unlabeled amino acids contributed to the carbon balance. Under anaerobic conditions, all the glucose carbons consumed could be accounted for solely by the NMR-identified products.

Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi.

The degradation of glucose by Trypanosoma cruzi leads to the excretion of succinate. Malate dehydrogenase (MDH) participates in this process by reducing to malate the oxaloacetate synthesized by the glycosomal enzyme, phosphoenolpyruvate carboxykinase. The best coupling for these two sequential reactions would be attained if both enzymes were placed in the same subcellular compartment. The intracellular distribution of the MDH activity in epimastigotes of T. cruzi was studied by two methods. Selective disruption of cellular membranes with increasing concentrations of digitonin, indicated that trypanosomal MDH is particulate. Isopycnic centrifugation in a sucrose gradient of a large granule fraction, obtained by grinding the cells with silicon carbide, showed the presence of two MDH activities: one banding together with the glycosomal marker phosphoenolpyruvate carboxykinase, the other with the mitochondrial marker citrate synthase. Isoelectrofocusing of cell-free extracts led to the separation of two enzyme forms, with pI values of about 3.5 (MDHa) and 9.4 (MDHb). These forms had similar molecular weights (approx. 60 000) and apparent Km values, but showed a small but consistent difference in their pH optima (9.23 for MDHa and 9.05 for MDHb), and in their activation by inorganic phosphate (apparent Ka values of 33 mM and 87 mM, for MDHa and MDHb, respectively). Determination of the pH optima of the enzyme forms separated by isopycnic centrifugation suggests that the glycosomal enzyme form is MDHa, and the mitochondrial one is MDHb.

Rhodoquinone requirement of the Hymenolepis diminuta mitochondrial electron transport system.

The occurrence of rhodoquinone as a mitochondrial membrane component was demonstrated in adult Hymenolepis diminuta. Chromatographic separation of pentane extracts, from lyophilized mitochondrial membranes, coupled with spectral analyses of separated material demonstrated the presence of rhodoquinone. The presence of ubiquinone was not apparent. Rhodoquinone content of membranes was about 1.2 micrograms (mg protein)-1. The rhodoquinone requirement of the H. diminuta electron transport system was demonstrated both in terms of the less active NADH oxidase and the physiologically required, NADH-dependent fumarate reductase employing lyophilized mitochondrial membranes as the source of activities. Pentane extraction of membranes virtually abolished the oxidase and fumarate reductase systems. Supplementation of pentane-treated membranes with H. diminuta rhodoquinone restored oxidase and fumarate reductase activities to levels simulating those of lyophilized membranes. Ubiquinone did not substitute for rhodoquinone. The rhodoquinone-reconstituted membranes displayed rotenone sensitivity. These findings represent the first direct demonstration of the rhodoquinone requirement of helminth electron transport-coupled oxidase and fumarate reductase.

Regulation of biosynthesis of guanidinosuccinic acid in isolated rat hepatocytes and in vivo.

To clarify the metabolic pathway of guanidinosuccinic acid (GSA), we investigated the relationship between GSA synthesis and the urea cycle. In isolated rat hepatocytes, GSA formation increased as urea concentration was raised; the effect of urea was not modified by the addition of ammonium chloride. Other urea cycle intermediates, including arginine and cyanate, a degradation product of urea, failed to stimulate GSA synthesis. Ornithine and arginine, which stimulate urea synthesis, strongly inhibited urea-stimulated GSA synthesis in the presence of 10 mM ammonium chloride, but the inhibitory effect of ornithine was not observed when ammonium chloride was not present. Citrulline (5 mM) strongly inhibited urea-stimulated GSA synthesis with or without ammonium chloride. D,L-norvaline, which inhibits urea cycle enzymes, strongly inhibited GSA synthesis. Following urea injection, hepatic GSA levels also increased in vivo, but there was little change in hepatic arginine. However, the addition of ornithine or D,L-norvaline inhibited the production of hepatic GSA, although arginine was increased substantially. These results indicate that GSA synthesis occurs in rat hepatocytes and is stimulated by urea. The data also suggest that the urea cycle enzymes catalyze some of the biochemical reactions in the GSA synthetic pathway.

The presence and possible function of methylmalonyl CoA mutase and propionyl CoA carboxylase in Spirometra mansonoides.

Both spargana and adult forms of Spirometra mansonoides were shown to accumulate lactate, succinate, acetate, and propionate upon in vitro incubation. Adults differed markedly from the spargana in that quantitatively the most significant products of the former were acetate and propionate while the latter formed primarily acetate and lactate. The adults accumulated approximately 32 times more propionate than the spargana per gram of tissue. In accord with this propionate formation, propionyl CoA carboxylase and methylmalonyl CoA mutase have been found to be present in both stages of the parasite. As might be predicted, however, the activities of the carboxylase and mutase were 100-fold and 10-fold higher, respectively, in the adults as compared to the larvae. A possible physiological relationship between propionate formation and energy generation is suggested. Accordingly, inorganic 32P was incorporated into ATP upon incubation of methylmalonyl CoA with a homogenate obtained from adult S. mansonoides. Since methylmalonyl CoA mutase requires vitamin B12 coenzyme, a relationship between vitamin B12 content and propionate formation in helminths is suggested.

[Mechanisms for improving the energy metabolism and function of the hypoxic myocardium with amino acids]. / Mekhanizmy uluchsheniia énergeticheskogo obmena i funktsii gipoksicheskogo miokarda aminokislotami.

The relationship between metabolism of the main myocardial amino acids, glutamate, aspartate and alanine, and energy state of hypoxic myocardium, was studied. Depression of cardiac contractile function during asphyxia in rats was accompanied by a decrease in glutamate mitochondrial and tissue contents and an increase in the tissue alanine and mitochondrial aspartate. The reduction of mitochondrial glutamate in asphyxia was related to losses of intramitochondrial ATP and state 3 respiration with glutamate and malate. Using NMR technique, exogenous glutamate and oxaloacetate were shown to increase succinate formation coupled with ATP and CTP production in the rat heart mitochondria in absence of aeration. These data suggest that glutamate and products of its transamination decrease the contraction of hypoxic myocardium stimulating anaerobic energy formation in the tricarboxylic acid cycle.