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Biomedical subjects

Y Anraku

Publications and source records attributed to Y Anraku.

At least 217 records · Page 12Linked to original sources
PubMed1977

Transport of sugars and amino acids in bacteria. XVIII. Properties of an isoleucine carrier in the cytoplasmic membrane vesicles of Escherichia coli.

The properties of the carrier for isoleucine in Escherichia coli were studied using cytoplasmic membrane vesicles (IM vesicles) prepared by the method of Yamato, Anraku, and Hirosawa (J. Biochem. 77, 705 (1975)). The IM vesicles exhibited respiration-dependent isoleucine transport activity which was more than 30-fold higher than that of "Kaback vesicles" prepared by our hand from the same strains of E. coli K12. The isoleucine carrier activity of IM vesicles was inhibited by norleucine but not by threonine. The carrier was driven by proton motive force. Mutants were isolated which had lost the carrier activity for isoleucine, as judged by assay with IM vesicles. Using these mutants, the effects of binding proteins specific for branched chain amino acids on the translocation of substrate in IM vesicles were studied. Leucine-isoleucine-valine-threonine-binding protein (LIVT-binding protein) stimulated the initial rate of isoleucine uptake by IM vesicles only when the vesicles possessed carrier activity and it did not affect the Kt value for entry of substrate. This evidence suggests the partial reconstitution of the osmotic shock-sensitive transport reaction in which the binding protein seems to affect the carrier activity with turnover ability.

Amino Acids↗
PubMed1976

Escherichia coli membrane D-lactate dehydrogenase. Isolation of the enzyme in aggregated from and its activation by Triton X-100 and phospholipids.

D-Lactate dehydrogenase was obtained in an aggregated form consisting of 2 to 3 molecules of the monomer enzyme after removal of most Triton X-100 from the preparation as described previously (1). The aggregate dissociated reversibly to the monomeric form after addition of 0.06% or 1.0% Triton X-100. Formation of these aggregates was confirmed by the finding that the enzyme activity was only partially sensitive to specific antibody. The specific activity of the aggregated enzyme was one-third that of the enzyme with Triton X-100 and it increased approximately 5-fold on addition of phospholipids or cardiolipin of Escherichia coli and lecithin from egg yolk. Both the monomer and micelle forms of Triton X-100 caused activation of the enzyme. The activity of the aggregates after preincubation with Triton X-100 or phospholipids was completely inhibited by specific antibody. The difference in the properties of the aggregated enzyme after preincubation with Triton X-100 and with phospholipids suggested that its interaction with phospholipids was stronger than with Triton X-100. Kinetic studies also suggested a difference between the interactions of the enzyme with phospholipids and with Triton X-100. Aggregated enzyme had an apparent Km value for D-lactate similar to that of membrane-bound enzyme after preincubation with phospholipids.

Cell Membrane↗
PubMed1975

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.

Adenosine Triphosphatases↗
PubMed1975

Transport of sugars and amino acids in bacteria. XVI. Theory and evaluation of a model for the membrane transport reaction mediated by a single carrier with three binding sites for substrate.

A novel model is presented for bacterial active transport reactions which show a curvilinear Eadie-Hofstee plot and negative homotropic cooperativity in the kinetics of substrate uptake. Various models of a single carrier with multi-binding sites for substrate were constructed and examined theoretically. The fit of these models with experimental data on the kinetics of branched chain amino acid transport reactions were tested by iterative computation using the non-linear least square method. The transport model which fitted the experimental data best consisted of a single carrier with three binding sites for substrate in which one of the substrate-carrier complexes, CSS, is not active in translocating substrate across the cytoplasmic membrane. The mechanism of homeostatic regulation of the intracellular concentration of amino acids by active transport systems is discussed on the basis of this transport model.

Binding Sites↗
PubMed1975

Transport of sugars and amino acids in bacteria. XIII. Mechanism of selective inhibition of the active transport reactions for proline, leucine, and succinate by zinc ions.

A mutant (S-1) of Escherichia coli which lacks succinate dehydrogenase [EC 1.3.99.1] but has normally succinate transport system, and various oxidase activities other than succinate, was isolated from the strain U24. Using these strains studies were made on the effects of zinc ions on the active transport reactions for succinate proline, and leucine. Zinc ions inhibited succinate transport reaction and succinate oxidation by intact cells. The active transport reaction for proline, which was supported by endogenous energy source in intact cells, was slightly inhibited by zinc ions. This inhibition was not reversed by large excess of magnesium ions. The initial rates of active uptakes of proline were increased by exogenous energy sources such as succinate and glycerol and zinc ions greatly inhibited these. However, D-lactate dependent proline uptake was enhanced slightly by the presence of zinc ions. It was found that zinc ions at a high concentration enhanced the steady level of proline accumulation in cells. This mechanism was studied in detail and we concluded that zinc ions inhibited completely the exchange-exit reaction for proline. Effects of zinc ions on the active transport reaction for leucine were rather simple when compared with these for proline. Zinc ions inhibited strongly the initial rates of leucine uptakes which were driven by endogenous and exogenous energy sources. The ions also inhibited the exchange-exit reaction.

Biological Transport, Active↗
PubMed1975

Transport of sugars and amino acids in bacteria. XV. Comparative studies on the effects of various energy poisons on the oxidative and phosphorylating activities and energy coupling reactions for the active transport systems for amino acids in E. coli.

The effects of various energy poisons on oxidation of respiratory substrate, synthesis of cellular ATP, and energy transformation reaction in intact Escherichia coli cells were studied systematically. Various mutants were, therefore, used in which specific functions in the energy-transducing reactions were defective or altered. The energy poisons examined were: sodium azide. DPPA and azidebenzenes which are inhibitors of respiratory-chain phosphorylation, SF6847, and CCCP which are known to be uncouplers, zinc sulfate which is an inhibitor for certain dehydrogenases, and sodium arsenate and sodium fluoride which are inhibitors of glycolytic synthesis of ATP. The preferential inhibitions occurred in the oxidation reactions with certain respiratory substrates by energy poisons used. DPPA inhibited glycerol oxidation much more strongly than succinate oxidation. However, DPPA could inhibit the oxidation of both glycerol 3-phosphate and succinate by membrane fraction strongly while the oxidation of NADH and D-lactate slightly. It inhibited glycerol 3-phosphate dehydrogenase [EC 1.1.2.1] strongly as well as succinate dehydrogenase [EC 1.3.99.1],.but not D-lactate dehydrogenase of membrane fraction. MAB and other azidebenzene derivatives inhibited succinate oxidation preferentially. SF6847 and CCCP inhibited succinate oxidation strongly, while sodium azide inhibited it weakly and these three poisons were less inhibitory for glycerol oxidation. DPPA, sodium azide, SF6847, and CCCP inhibited the synthesis of ATP coupled with respiration but not with glycolysis. Zinc sulfate inhibited the cellular ATP synthesis coupled with either respiration or glycolysis.

Adenosine Triphosphate↗
PubMed1975

Transport of sugars and amino acids in bacteria. XIV. Preferential inhibition of oxidase activities and active transport reactions for amino acids by azidebenzenes.

4-Methylazidebenzene and various azidebenzene derivatives were prepared, and the effects of these compounds on oxidase activities and active transport reactions for amino acids in Escherichia coli cells were studied. Azidebenzenes inhibited succinate oxidation by intact cells preferentially to glycerol oxidation. However, the azidebenzenes could not inhibit succinate oxidation which was not coupled to phosphorylation. The compounds inhibited succinate driven proline uptake much more strongly than isoleucine uptake. Unlike sodium azide and diphenyl phosphorazidate, azidebenzenes did not inhibit membrane-bound, Mg2+-requiring ATPase [EC 3.6.1.3] of E. coli. Reactivities of various azide compounds in the mechanism of inhibition for energy transducing and energy transforming reactions were discussed briefly.

Adenosine Triphosphatases↗