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Biomedical subjects

E Kun

Publications and source records attributed to E Kun.

At least 91 records · Page 5Linked to original sources
PubMed1978

Ethanol oxidation by isolated rat-liver cells. Stimulatory effects of fructose.

Isolated liver cells from starved or fed rats were used to examine the mechanisms by which fructose stimulates hepatic ethanol oxidation, particularly in relation to the role of intercompartmental hydrogen transfer. The presence of added fructose stimulated ethanol oxidation, the degree of stimulation being directly related to the initial fructose concentration up to a level of 10--20 mM. In the presence of fructose and ethanol, sorbitol, glycerol and glycerol 3-phosphate accumulated. The stimulation of ethanol oxidation by fructose could be attributed to its ability to promote the oxidation of cytoplasmic NADH by the provision both of cytoplasmic hydrogen sinks and components of the glycerol 3-phosphate shuttle. Shuttles mediated by malate dehydrogenase were unimportant for fructose-stimulated ethanol oxidation. Rates of respiration and gluconeogenesis were similar over a fructose range of 5--20 mM although ethanol oxidation was faster at higher fructose concentrations. Hence the stimulation of ethanol oxidation by fructose is not merely a direct function of the energy demands of gluconeogenesis.

Animals↗
PubMed1978

Analysis of larger than tetrameric poly(adenosine diphosphoribose) by a radioimmunoassay in nuclei separated in organic solvents.

Anitibodies were prepared against poly(adenosine diphosphoribose) of an average chain length of 40 adenosine diphosphoribose units by repeated injection of the polymer mixed with methylated albumin and adjuvants into rabbits. The antibody was present mainly in the 7 S fraction of the immunoglobulins. A membrane binding assay was developed, and its specificity determined for the detection of (adenosine diphosphoribose)ngreater than4 in organs. The method is suitable for the study of the variation of the polymer content of nuclei. The size recognition of the anti-poly(adenosine diphosphoribose) globulin fraction was the same for polymers composed of 4--40 adenosine diphosphoribose units, but smaller oligomers were not detectible. A quantitative extraction technique was developed and applied for radioimmunoassay of nuclear (adenosine diphosphoribose)n greater than 4. Organs were freeze-clamped, freeze dried, broken into subcellular fragments in a colloid mill, and the nuclear fraction was subsequently separated in organic solvents in order to preserve the polymer. Nicotinamide and nicotinic acid, when administered in vivo, augmented the (adenosine diphosphoribose)n greater than 4 content of rat liver and heart. Tissues of infant pigeons contained larger quantites of (adenosine diphosphoribose)ngreater than4 than tissues of adult rats.

Animals↗
PubMed1978

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates.

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H+). Although its acid-base properties were indistinguishable from those of epsilon-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the epsilon-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0. It is apparent that the assay of epsilon-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236--250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.

Acyl Coenzyme A↗
PubMed1977

Enzymatic formation of glutathione-citryl thioester by a mitochondrial system and its inhibition by (-)erythrofluorocitrate.

A soluble extract of the mitochondrial compartment composed of the inner membrane and matrix catalyzes the enzymatic synthesis and hydrolysis of the 1:1 adduct of citric acid and glutathione. The adduct was identified as the thioester by isolation with single and double isotope labeling ([(14)C]citric acid and [(35)S]glutathione) and by conversion to the monohydroxamate of citric acid and comparison with the synthetic product by thin layer chromatography and high voltage electrophoresis. The enzymatic formation of the thioester (pH optimum 7.39 at 30 degrees ) requires oxidized glutathione and citrate; both substrates exhibit a Michaelis-Menten kinetics. During the enzymatic reaction equimolar quantities of thioester and glutathione sulfinic acid are formed. After gel filtration or salt fractionation the enzyme system requires Mn(2+) (or Mg(2+), which is less effective) for maximal activity. When extracts of mitoplast are tested, the time course of reaction is biphasic due to the rapid synthesis of the product by the thioester-forming system (molecular weight 171,000) followed by its decay by the hydrolase (molecular weight 71,000). The two systems were separated by molecular filtration on Sephadex G-200 and by precipitation with (NH(4))(2)SO(4). The thioester-forming system is inhibited by preincubation with 0.5 mM mersalyl. Other inhibitors are 1,2,3-propane tricarboxylic acid, 10 mM Ca(2+), 200 mM K(+), and the free radical trapping agent, phenazine methosulfate. The citrate-glutathione thioester formation is irreversibly and specifically inhibited by (-)erythrofluorocitrate (50% inhibition at 25 pmol of added fluorocitrate per mg of protein), which forms a trichloroacetic acid-stable adduct with the enzyme protein (at 50% inhibition, 0.8 pmol is bound to 1 mg of protein). Synthesis of malyl-glutathione thioester by inner membrane vesicles is selectively inhibited by (-)erythrofluoromalate.

Animals↗
PubMed1976

Kinetics of ATP-dependent Mg2+ flux in mitochondria.

ATP-dependent Mg2+ accumulation in isolated mitochondria occurs predominantly in the matrix and inner membrane compartments. In mitochondria contaminated with lysosomes, the time course and magnitude of ATP-dependent Mg2+ accumulation are influenced by various cytoplasmic substances, besides substrates of the citric acid cycle. Removal of lysosomes by treatment of the mitochondrial preparation with low concentrations of digitonin, which does not damage the mitoplast, eliminates the modifying influence of cytoplasmic components on Mg2+ flux. In lysosome-free mitochondria, the kinetics of Mg2+ flux is dependent only on the concentration of ATP, of Mg2+, and on the availability of site specific reducing substrates of the electron transport system. Oligomycin at concentrations sufficient to inhibit phosphorylation coupled electron transport and ATP synthesis does not modify Mg2+ flux, which is dependent on added ATP. Site specific inhibitors of the electron transport system inhibit the augmenting effect of oxidizable substrates on Mg2+ uptake, even when electron transfer is inhibited by oligomycin. Atractyloside, by inhibiting the action of externally added ATP, diminishes Mg2+ flux. Ruthenium red is a powerful inhibitor of ATP dependent Mg2+ flux. Uncouplers not only inhibit Mg2+ uptake, but induce Mg2+ efflux. From the time course of Mg2+ flux, a first-order rate constant of egress of Mg2+ and other kinetic constants were calculated and a kinetic model was derived which describes the bi-directional movement of Mg 2+ in mitoplasts.

Adenosine Triphosphate↗
PubMed1976

Covalent modification of proteins by metabolites of NAD+.

Covalently bound adducts of ply(L-lysine), bovine serum albumin, lysine rich histone (f1) and deoxyribonucleotidase I (DNase, EC 3.1.4.5) with adenosine diphosphoribose and ribose-5-phosphate were prepared at pH 7.4 and 9.5. Macromolecular adducts of bovine serum albumin and histone (f1) were isolated by gel filtration and electrophoresis. Reduction of products by NaBH4 did not dissociate the ribose-5-phosphate moiety from macromolecules. Specific introduction of 3H into the adducts also indicated Schiff base formation. The reaction of ribose-5-phosphate with epsilon-amino groups of histone (f1) approached 70-90% saturation. Spermine and spermidine also react with adenosine diphosphoribose and ribose-5-phosphate to form 1:1 Schiff bases. It is proposed that high turnover of cellular NAD+ is the source of aldehydic metabolites which may regulate macromolecular metabolism by covalent modification of nuclear proteins, whereas polyamines serve as modulators of this control cycle.

Adenosine Diphosphate Sugars↗
PubMed1975

Macromolecular enzymatic product of NAD+ in liver mitochondria.

Rat liver mitochondria contain a Mg2+-requiring system that transfers the ADP-ribose moiety of NAD+ to an acceptor protein. The enzyme system was extracted in a soluble form and the ADP-ribosylated protein product was isolated by hydroxyapatite and Sephadex chromatography. The ADP-ribosylated protein product has a molecular weight of 100,000 and can be dissociated into subunits of 50,000 daltons by sodium dodecyl sulfate gel electrophoresis. Incubation of the isotopically labeled ADP-ribosylated protein with nicotinamide and a mitochondrial extract yields labeled NAD+, indicating apparent reversibility of the reaction. Enzymatic degradation of the ADP-ribosylated protein with snake venom phosphodiesterase liberates AMP and ADP-ribose or its isomer. Identification of these products and reversibility of the reaction show that the ADP-ribose moiety of NAD+ is the molecular species that is transferred to the acceptor protein. A fraction of the protein-bound ADP-ribose appears to be present as an an oligomer. The enzymatic protein-ADP-ribosylating reaction is inhibited by nicotinamide, ADP-ribose, the fluorophosphate of AMP, and picrylsulfonic acid.

Adenosine Diphosphate Sugars↗
PubMed1974

Effects of bicarbonate on intercompartmental reducing-equivalent translocation in isolated parenchymal cells from rat liver.

1. Incubation of isolated liver cells in a medium containing bicarbonate raises malate concentrations almost sixfold compared with values obtained in a bicarbonate-free phosphate medium. The malate concentration of about 0.3mm in bicarbonate medium is of the same order as the K(m) for malate dehydrogenase. 2. The utilization of ethanol, glyercol and sorbitol was increased by 20-35% in bicarbonate medium. 3. Fluoromalate, a specific inhibitor of malate dehydrogenase and the malate carrier, inhibited or ethanol oxidation by 23%, glycerol uptake by 20% and sorbitol uptake by 42% in bicarbonate medium, but had a much smaller inhibitory action in phosphate medium. In consequence fluoromalate almost abolished the stimulatory effects of bicarbonate on substrate utilization. 4. Difluoro-oxaloacetate, a specific inhibitor of aspartate aminotransferase, had about one-half the inhibitory activity of fluoromalate. The two inhibitors in combination were less effective than fluoromalate by itself. 5. It is concluded that bicarbonate stimulates the utilization of reduced substrates, which are oxidized in the cytoplasmic compartment of the liver cell, by increasing the activity of rate-limiting malate dehydrogenase-dependent intercompartmental hydrogen shuttles. Both malate-oxaloacetate and malate-aspartate systems are involved in these hydrogen-translocation processes.

Animals↗