Biomedical subjects

Y Anraku

Publications and source records attributed to Y Anraku.

At least 181 records · Page 10Linked to original sources

PubMed1985

Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae.

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.

Adenosine Triphosphate↗

PubMed1985

Transforming genes in human leukemia cells.

High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay.

Autoradiography↗

PubMed1985

Quantitative determination of cytochromes in the aerobic respiratory chain of Escherichia coli by high-performance liquid chromatography and its application to analysis of mitochondrial cytochromes.

A method was established for quantitative determination of the composition and the amounts of cytochromes in the aerobic respiratory chain of Escherichia coli. Cytochromes were solubilized with 3%(W/V) Sarkosyl and the extract was analyzed by high-performance liquid chromatography on a gel permeation column of TSK gel-G3000SW. This analytical system required only about 0.5 mg of membrane protein and 20 min per assay. The effects of the gene dosage of F-prime factors and plasmids on cytochromes were analyzed with this system, and the system was applied to the analysis of mitochondrial cytochromes.

Aerobiosis↗

PubMed1984

Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae. Evidence of seven independent proton/amino acid antiport systems.

The substrate specificities of the amino acid transport systems of vacuoles of the yeast, Saccharomyces cerevisiae, were investigated using purified vacuolar-membrane vesicles (Ohsumi, Y., and Anraku, Y. (1981) J. Biol. Chem. 256, 2079-2082). Ten amino acids: arginine, lysine, histidine, phenylalanine, tryptophan, tyrosine, glutamine, asparagine, isoleucine, and leucine, were taken up actively into the vesicles. Kinetic studies indicated the presence of seven independent H+/amino acid antiport systems with narrow substrate specificity, which were all driven by a proton motive force established by ATP hydrolysis. The Kt and Vmax values, and the specific inhibitors for the arginine, arginine-lysine, histidine, phenylalanine-tryptophan, tyrosine, glutamine-asparagine, and isoleucine-leucine transport systems were determined.

Amino Acids↗

PubMed1984

An arginine/histidine exchange transport system in vacuolar-membrane vesicles of Saccharomyces cerevisiae.

High-affinity arginine-uptake activity in vacuolar-membrane vesicles from the yeast Saccharomyces cerevisiae was detected in the presence of ATP and histidine. Histidine increased the initial rate of uptake and the steady level of accumulation of arginine. When the vesicles were preloaded with histidine in the absence of ATP and diluted, transient uptake of arginine took place with concomitant downhill efflux of histidine from the vesicles. Kinetic examination indicated the existence of a unique transport system which catalyzed exchange of 1 mol of arginine outside with 1 mol of histidine inside the vacuolar membrane. The Ktentry for arginine and the Ktexit for histidine were determined to be 0.1 and 1.1 mM, respectively.

Arginine↗

PubMed1984

Mechanism of proline transport in Escherichia coli K12. I. Effect of a membrane potential on the kinetics of 2H+/proline symport in cytoplasmic membrane vesicles.

The stoichiometric coupling mechanism of the membrane potential (delta psi) in the reaction of H+/proline symport was investigated kinetically, using cytoplasmic membrane vesicles of the proline carrier-overproducing strain of Escherichia coli MinS/ pLC4 -45. When a delta psi was imposed across the cytoplasmic membrane by respiration, the Michaelis constant of transport (Kt) was lowered to about 1 microM, which was 2 orders of magnitude smaller than that of passive influx and efflux, and the maximum velocity (Vmax) was concomitantly enhanced as an exponential function of delta psi. Thermodynamically, the carrier translocated proline with a stoichiometry of 2 mol of protons versus 1 mol of substrate when driven by a delta psi at pH 8.0. Data on the delta psi dependence of Vmax of proline transport could be explained quantitatively by the Geck-Heinz hypothesis (Geck, P., and Heinz, E. (1976) Biochim, Biophys. Acta 443, 49-63). A symmetrical model of the 2H+/proline symport via formation of a carrier/H+/substrate (CH+H+S) intermediate is proposed. In this model, the effect of delta psi on the Kt was resolved as stimulation of formation of a transport intermediate, whereas the effect of delta psi on the Vmax was explained by enhancement of translocation of loaded carriers between the two sides of the membrane.

Biological Transport↗

PubMed1984

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles.

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).

Amino Acid Transport Systems, Neutral↗

PubMed1984

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier.

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.

Amino Acid Transport Systems, Neutral↗

PubMed1984

Terminal oxidases of Escherichia coli aerobic respiratory chain. II. Purification and properties of cytochrome b558-d complex from cells grown with limited oxygen and evidence of branched electron-carrying systems.

Cytochrome b558-d complex, a terminal oxidase in the respiratory chain of Escherichia coli K12 grown under the condition of limited oxygen, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides, with Mr = 26,000 and 51,000, determined with gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 12.3 nmol of protoheme, 9.54 nmol of cytochrome d, and 26.6 nmol of iron/mg of protein. The enzyme is a "cytochrome bd-type oxidase," which shows absorption peaks at 558 and 624 nm in the difference spectrum at 77 K. This oxidase combines with CO, and the Soret region of the CO difference spectrum at room temperature has a peak at 420 nm and troughs at 430 and 442 nm. The oxidation-reduction potentials of cytochrome b558 and cytochrome d at pH 7.4 were estimated to be +10 mV and +240 mV, respectively. The cytochrome b558-d complex catalyzes the oxidation of ubiquinol-1, menadiol, and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity was inhibited by the respiratory inhibitors piericidin A, KCN, and NaN3, but the cytochrome b558-d complex was less sensitive to the inhibitors than the cytochrome b562-o complex. The Km values for oxygen of purified cytochrome b558-d complex and cytochrome b562-o complex were determined to be 0.38 and 2.9 microM, respectively. Formation of a membrane potential by the reconstituted cytochrome b558-d complex in liposomes was observed with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide on addition of ubiquinol-1. This is the first indication that there is a coupling site in the terminal oxidase, which contains cytochrome d. Steady state kinetics of cytochromes in the membrane showed that these oxidase complexes branch at the site of ubiquinone-8 in the respiratory chain. From these and previous results, branched electron-carrying systems of the E. coli respiratory chain are proposed.

Aerobiosis↗

PubMed1984

Terminal oxidases of Escherichia coli aerobic respiratory chain. I. Purification and properties of cytochrome b562-o complex from cells in the early exponential phase of aerobic growth.

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli K12, was isolated in a highly purified form. The purified oxidase is composed of equimolar amounts of two polypeptides, with Mr = 33,000 and 55,000, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 19.5 nmol of heme and 16.8 nmol of copper/mg of protein, but no detectable nonheme iron, phospholipid, ubiquinone, or menaquinone. In the difference spectrum at room temperature, the oxidase shows a single alpha absorption peak at 560 nm and at 77 K it shows two alpha absorption peaks at 555 and 562 nm. This oxidase combines with CO and the CO difference spectrum at room temperature has a peak at 416 nm and a trough at 430 nm in the Soret region. Its oxidation-reduction potential is estimated to be 125 mV (pH 7.4) and it is pH-dependent (-60 mV/pH) in medium of pH 6.0 to 7.4. It catalyzes electron transport to oxygen via ubiquinol and ascorbate in the presence of phenazine methosulfate or N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity depends on phospholipids and is sensitive to respiratory inhibitors, such as 2-heptyl-4-hydroxyquinoline N-oxide, piericidin A, KCN and NaN3. The divalent cations Zn2+, Cd2+, and Co2+ inhibit the oxidase activity extensively. The oxidase activity of the cytochrome b562-o complex was inhibited by photoinactivation with rose bengal, suggesting that the inhibition by zinc ion results from modification of a histidine residue of cytochrome o.

Aerobiosis↗

PubMed1984

Cloning of cybB, the gene for cytochrome b561 of Escherichia coli K12.

A 37 kb fragment of DNA from an F-prime factor, F100-12, which showed a gene dosage effect on b-type cytochromes, was cloned with a cosmid vector, pHC79. Gel filtration of cytochromes and product analysis of the hybrid plasmids indicated that this fragment contained cybB, the structural gene for cytochrome b561. A chromosomal DNA fragment carrying the cybB gene was cloned by the plaque hybridization technique with Charon 4A as a vector. The gene was subcloned into pBR322 and was located in a 1.3 kb DNA fragment. It was concluded that the cybB gene is located on the chromosome of Escherichia coli K12.

Chromosome Mapping↗

PubMed1984

Chromosomal location of the Escherichia coli cytochrome b556 gene, cybA.

The amounts of cytochrome b556 in the cytoplasmic membranes of several Escherichia coli K12 strains having F-prime factors and a lambda transducing phage were determined. The amount was amplified about two-fold in strains having F100-12 and F152, but not in strains having F100-11, F8 and lambda psu+2glnS+. The strain TK3D11, which lacks the kdp-gltA region (deletion D-01) of the E. coli chromosome, did not synthesize cytochrome b556 at all. From these results, the gene cybA encoding cytochrome b556 was located in the kdp-gltA region. In the cytochrome b556-deficient mutant, a novel b type cytochrome, cytochrome b561 which is a product of the gene cybB, was identified. It seems to function as a physiological electron transferring cytochrome in place of cytochrome b556 in this mutant.

Bacteriophage lambda↗

PubMed1984

Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants.

A cal1-1 mutant of the yeast Saccharomyces cerevisiae showing Ca2+-dependent growth was isolated. Its growth continued exponentially in Ca2+-rich medium, but stopped in Ca2+-poor medium at 37 degrees C. Mg2+ ions could not replace Ca2+ ions. In Ca2+-poor medium, the mutant cells stopped growing homogeneously at the stage of cell division cycle with a tiny bud. The nucleus in these arrested cells was in the G2 stage, judging from observation after nuclear staining and determination of the DNA content. Trifluoperazine-dependent pseudorevertants, which could grow in the presence of 20 microM to 80 microM trifluoperazine in Ca2+-poor medium at 37 degrees C, were obtained from this cal1-1 mutant. The suppressor mutation, tfr1, itself conferred trifluoperazine resistance. Other calmodulin inhibitors structurally unrelated to trifluoperazine had similar effects to trifluoperazine on these pseudorevertants. These results suggest that Ca2+ ions and a calmodulin play important roles in the yeast cell division cycle at the stage of bud growth and nuclear division.

Calcium↗

PubMed1983

Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae.

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.

Adenosine Triphosphate↗

PubMed1983

Mechanism of glutamate transport in Escherichia coli B. 1. Proton-dependent and sodium ion dependent binding of glutamate to a glutamate carrier in the cytoplasmic membrane.

Specific binding of glutamate to its carrier was investigated by using cytoplasmic membrane vesicles prepared from Escherichia coli B. The binding activity was specifically affected by the Na+ and H+ concentrations of the medium. Cytoplasmic membrane vesicles from the mutant strain 36-39 that is defective in the Na+-dependent glutamate transport system showed no binding of glutamate. Addition of the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile or carbonyl cyanide m-chlorophenylhydrazone, or the ionophore monensin or nigericin, did not inhibit the binding, indicating that the binding reaction is not energy dependent. The parameters of binding were determined in reaction media with various combinations of H+ and Na+ concentrations. The maximum number of binding sites was constant and determined to be 70 pmol/mg of membrane protein, irrespective of the concentrations of H+ and Na+ in the medium. The apparent dissociation constant, however, was greatly affected by changes in the concentrations of both H+ and Na+, in such a way that it was expressed by a linear combination of the reciprocals of the H+ and Na+ concentrations. The characteristics of binding can be explained best by supposing that glutamate can bind only to a H+/Na+/carrier complex that is formed by random binding of H+ and Na+ to the unloaded carrier. The physiological role of this elementary binding reaction and of this quaternary complex as an active intermediate in the process of glutamate transport is discussed.

Biological Transport↗

PubMed1983

Mechanism of glutamate transport in Escherichia coli B. 2. Kinetics of glutamate transport driven by artificially imposed proton and sodium ion gradients across the cytoplasmic membrane.

Simultaneous imposition of a pH gradient (delta pH, interior alkaline) and a sodium gradient (delta pNa, [Na+]out greater than [Na+]in) across cytoplasmic membrane vesicles from Escherichia coli B led to a several hundred fold accumulation of glutamate. Although less effective, delta pH (interior alkaline)( alone caused accumulation of glutamate in the presence of Na+. In addition, delta pNa ([Na+]out greater than [Na+]in) alone also drove the transport system, where the maximum level of glutamate accumulation was affected by the pH of the medium. A membrane potential imposed by valinomycin-induced K+ diffusion (interior negative) enhanced the accumulation, indicating that the system operation in an electrogenic manner. The Michaelis constant of glutamate transport was greatly affected by changes in the concentrations of both Na+ and H+ and could be expressed by a linear combination of the reciprocals of the Na+ and H+ concentrations in the medium. On the contrary, a membrane potential (interior negative) exerted its effect by increasing the maximum velocity. When membrane vesicles were loaded with glutamate and Na+, but not with glutamate alone, rapid efflux of glutamate with Na+ as the cocation down the concentration gradients took place upon dilution. These results indicate that both Na+ and H+ are syn-coupled ions of glutamate transport in E. coli B and that the carrier/Na+/H+/Glu- complex observed in the binding reaction is an intermediate in the transport.

Biological Transport↗

PubMed1982

Random distribution of the glucose transporter of human erythrocytes in reconstituted liposomes.

The glucose transporter of human erythrocytes was reconstituted with soybean phospholipids by the freeze-thaw/sonication method and the distribution of the transporter molecules in liposomes was studied. The steady state level of glucose transport in reconstituted liposomes showed saturation when increased amounts of the transporter were used for reconstitution. The saturation curve fitted well to a theoretical curve which was derived assuming a Poisson distribution of the transporter. Freeze-fracture electron micrographs showed random distribution of intramembraneous particles on liposomes, irrespective of liposome size or amount of the transporter added. A detailed study showed a parameter of the distribution (the ratio of transporter to liposome) obtained from the transport measurement can be used for the analysis of the distribution of intramembraneous particles, indicating that most of the molecules seen as particles were active in transport.

Blood Glucose↗

PubMed1982

Formation of a membrane potential by reconstructed liposomes made with cytochrome b562-o complex, a terminal oxidase of Escherichia coli K12.

A terminal oxidase of the Escherichia coli K12 respiratory chain (cytochrome b562-o complex) was reconstituted into liposomes by freeze-thaw/sonication method. Formation of a membrane potential (-145 mV) by the reconstituted cytochrome b562-o complex was observed with the fluorescent dye 3,3'-dipropylthiodicarbocyanine iodide on addition of an artificial electron donor ubiquinol-1 or ascorbate-phenazine methosulfate. The membrane potential formed was inhibited by the protonophore uncouplers 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and carbonylcyanide m-chlorophenylhydrazone, and the inhibitors of the oxidase system zinc sulfate, potassium cyanide, and 2-n-heptyl-4-hydroxyquinoline-N-oxide. This is the first indication that there is a coupling site in an E. coli terminal oxidase, which consists of b-type cytochromes.

Biological Transport↗