Oxidative metabolism of funicular tissue. I. Effect of exogenous substrates on oxygen consumption of funicular tissue in vitro]

Umbilical cord slices were incubated with various concentrations of either glucose, pyruvate or succinate. Furthermore the conversion of pyruvate to AcCoA and the oxidation of the latter were assessed respectively by measuring labelled CO2 evolved from 14C-pyruvate labelled on C1 or C2. The results suggest that 1) ADP stores are low since glucose induces a Crabtree effect; 2) the "succinoxidase" complex is far from being saturated and cannot account for the low oxidations observed under basal conditions; and 3) that the limiting step should be looked for either in Krebs' cycle or at the level of Complex I of the electron transport chain.     

The activity of Krebs cycle enzymes in the visual analyzer of normal rats and under stress]

In the visual analyzer of the intact animals higher activity of NAD-dependent dehydrogenases of tricarbonic acids cycle is observed in the retina and of FAD-dependent dehydrogenases in the occipital brain lobes. The influence of stress by Desiderato method elicits compensatory increase of the succinatedegidrogenase activity. The acute stress elicits a change of regulation of the activity of dehydrogenase of tricarbonic acids cycle, estimated by the reaction to functional load. Animals staying in the darkness after stress promotes restoration of the tricarbonic acids cycle of the enzymes activity up to the normal level.     

A new spectrophotometric assay for enzymes of purine metabolism. II. Determination of guanase activity.

A new method for the determination of guanase is described. Xanthine, the product of the guanase reaction, is oxidized by xanthine oxidase, forming uric acid and hydrogen peroxide. Hydrogen peroxide is further reduced to water by catalase in the presence of ethanol. The acetaldehyde formed in this reaction step is dehydrogenated NAD or NADP dependent by aldehyde dehydrogenase. The NADH or NADPH production is measured and utilized for the calculation of the guanase activity. The sensitivity of the method can be doubled by the addition of uricase, which oxidizes uric acid to permit the formation of another mole of hydrogen peroxide.     

Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria

Sonic oscillation of mitochondria usually leads to the release of a number of Krebs tricarboxylic acid cycle enzymes. These enzymes have, therefore, been referred to as soluble matrix enzymes. In the present report, we show that gentle sonic or osmotic disruption can be used to obtain a mitochondrial preparation where these enzymes appear to be organized in a large complex of proteins. Using citrate synthase as a marker for these enzymes, we show that the proposed complex is easily sedimented at 32,000 X g in 30 min. The exposed citrate synthase in these complexes can be inhibited by its antibody, indicating that the enzymes are not merely entrapped in substrate-permeable vesicles. The effects of pH, temperature, ionic strength, and several metabolites on the ability to obtain the sedimentable citrate synthase have been tested. These studies indicate that the complex is stable at conditions presumed to exist in situ. Electron microscopic studies show that gentle sonic oscillation gives rise to an efflux of mitochondrial matrix contents which tend to remain attached to the original membranes. The sedimentable fraction also contained four other presumably soluble Krebs tricarboxylic acid cycle enzymes: aconitase, NAD+-isocitrate dehydrogenase, fumarase, and malate dehydrogenase.     

Noninvasive tracing of Krebs cycle metabolism in liver

To quantify intrahepatic Krebs cycle metabolism, phenyl acetate, excreted in urine as a glutamine conjugate, was given to healthy subjects infused with [3-14C]lactate. They were studied after 60 h of fasting and when given glucose after an overnight fast. Distributions of 14C in glutamate from urinary phenylacetylglutamine and blood glucose were determined. Corrections to the distributions because of the fixation of 14CO2 formed from the [3-14C]lactate were determined by administering [14C]bicarbonate. Comparisons of distributions in glucose and glutamate support the assumption that the glutamate distributions reflect those in hepatic alpha-ketoglutarate. From the distributions in glutamate, the extent of exchange of labeled with unlabeled carbons and relative flow rates in the cycle in liver were estimated. Dilution of 14C by 12C in the cycle was found in the fasted but not the fed state. In the fasted state, pyruvate carboxylation was estimated to be at least twice the rate of Krebs cycle flux and the rate of pyruvate's decarboxylation less than 1/25 the rate of its carboxylation. In the fed state, the rate of decarboxylation was estimated to be between one-sixth and one-half the rate of carboxylation. The rate of conversion of oxalacetate to fumarate in both states appeared to be greater than 6 times the rate of Krebs cycle flux.     

Activity of Krebs cycle dehydrogenase and tissue respiration enzymes in cerebral hemispheres of rats under stress

It is established that emotional-painful stress and myocardium necrosis evoked in animals after the emotional-painful stress is accompanied by a disturbance in the creative metabolism and partial inhibition of cytochrome oxidase in cerebral hemispheres. A conclusion is made that the found changes may be one of the pathogenetic factors in formation of the neurosis-like states under stress and diseases originated from the effect of the emotional-psychic overstrain.     

Oxidative enzymes in the development of Fasciola hepatica L. II. Dehydrogenase activity of miracidium.

Histochemical methods were used to detect succinate, isocitrate, malate, lactate, alpha-glycerol phosphate' L-glutamate, alcohol, glucose-6-phosphate, 6-phosphogluconate, and beta-hydroxybutyrate dehydrogenases in miracidium--a free living larva of Fasciola hepatica. Examinations were carried out on miracidia which had just left egg covers and on those which had survived for 18 and 24 hours, respectively. Differences were observed regarding the occurrence and intensity of the enzyme activity depending on the age of the larvae. On the basis of the localization of dehydrogenases and persistence of their activity in the miracidia in various periods of their lives, the author concludes that in young larvae there exist three parallel modes of oxidation: glycolytic, in the tricarboxylic acid cycle, and in the pentose cycle. Moreover, there occur processes of amino acid metabolism and of fatty acid oxidation. The 18-hour larvae exhibit a marked predominance of oxidation in the Krebs cycle, the remaining modes being less pronounced, and after 24 hours the only way to obtain energy in the miracidium is oxidation in the Krebs cycle. Likewise noteworthy is the enhancement of the processes of fatty acid oxidation, this being evidenced by an intensified activity of beta-hydroxybutyrate dehydrogenase. The role of each enzyme in the metabolism of the larva is discussed.     

Integration of lipid utilization with Krebs cycle activity in muscle.

This essay illustrates the ways in which beta-oxidation and the citric acid cycle interact. These included: 1) competition for CoASH, 2) competition for NAD+, and 3) competition for FADH2 oxidation. By means of the above, the cell is able to maintain a precise coordination between the activation of fatty acids in the cytosol, beta-oxidation in the mitochondria, and the complete oxidation of acetyl-CoA to CO2 via the citric acid cycle throughout a wide range of energy demands and oxygen availability.