Yasuhiro ANRAKU

Publications (8)1.81 Total impact

• Article: Mechanism of Na+/proline symport inEscherichia coli: Reappraisal of the effect of cation binding to the Na+/proline symport carrier

  Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier inEscherichia coli were investigated in detail. The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain (PT21/pTMP5) was absolutely dependent on the presence of Na+, but did not necessarily require protonation of the carrier, in contrast to the model previously reported (Mogi, T., Anraku, Y. 1984.J. Biol. Chem. 259:7797–7801). Based on this and previous observations, we propose a modified model of the proline binding reaction of the proline carrier, in which a proton is supposed to be a regulatory factor for the binding activity. The apparent Michaelis constant of proline (Kt) of the transport activity of cytoplasmic membrane vesicles from the wild typeE. coli strain driven by a respiratory substrate, ascorbate, showed dependence on a low concentration of sodium ion. The Michaelis constant of sodium ion for transport (Kt Na) was estimated to be 25 m. The proline transport activities in membrane vesicles and intact cells were modulated by H+ concentration, the inhibitory effect of protons (pK a 6) being similar to that observed previously (Mogi, T., Anraku, Y. 1984.J. Biol. Chem. 259:7802–7806). Based on these observations and the modified model of substrate binding to the proline carrier, a model of the proline/Na+ symport mechanism is proposed, in which a proton is postulated to be a regulatory factor of the transport activity.
  Journal of Membrane Biology 02/1990; 114(2):143-151. · 1.81 Impact Factor
• Article: Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

  Toshifumi Nakao, Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putP was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the Hpal site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.
  MGG - Molecular and General Genetics 10/1988; 214(3):379-388.
• Article: Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

  Hiro Nakamura, Hiroshi Murakami, Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b 561 and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b 561 is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b 561 with those of other bacterial b-type cytochromes was observed.
  MGG - Molecular and General Genetics 03/1988; 212(1):1-5.
• Article: Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12

  Toshifumi Nakao, Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: The nucleotide sequence of the putP gene coding for the proline carrier in Escherichia coli has been determined and the amino acid sequence of the proline carrier deduced from it. The proline carrier is predicted to consist of 502 amino acids, resulting in a molecular weight of 54,343. The predicted protein is very hydrophobic (70% nonpolar amino acids), and its hydropathy profile suggests that it is composed of 12 hydrophobic segments with a mean length of 24.4 residues/segment. If these segments are assumed to be -helical, the mean length of each domain corresponds to the thickness of the hydrophobic core of the membrane. Potential promoter, catabolite gene activator protein (CAP) binding sites and several palindromic sequence, which might be regulatory regions by the putA gene product, were also found in the 5 flanking region of the postulated putP gene. A typical p-independent transcription termination signal was found after the terminator codon of the putP gene.
  MGG - Molecular and General Genetics 05/1987; 208(1):70-75.
• Article: Nucleotide sequence of putC, the regulatory region for the put regulon of Escherichia coli K12

  Toshifumi Nakao, Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: The nucleotide sequence of the putC region, from which divergent transcription of the putP and putA genes starts, was determined. The promoter region for the putA gene was restricted to the location between the HindIII site and the NcoI site or at the NcoI site by using putA-lacZ fusion plasmids and the transcriptional start for the putA gene was identified in the region between the HindIII site and the NcoI site by S1 mapping. This region also contains a potential CAP binding site, a ribosome binding site, and a sequence that is highly homologous to argTr. five potential promoters (putPp1-putPp5) for the putP gene, which were separate from the promoter region for the putA gene, were indicated by S1 mapping analysis of the putP gene transcripts. We concluded that the putC region is 419 bp long and contains two independent sets of promoters, regulating the expression of putP and putA genes in opposite directions. In addition, this region was found to contain an open reading frame (orf) capable of encoding a polypeptide of 111 amino acids in overlapping fashion. But studies using an orf-lacZ fusion gene showed that this open reading frame was not expressed.
  MGG - Molecular and General Genetics 01/1987; 210(2):364-368.
• Article: Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12

  Tatsushi Mogi, Hiroshi Yamamoto, Toshifumi Nakao, Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.
  MGG - Molecular and General Genetics 12/1985; 202(1):35-41.
• Article: Cytoplasmic Membrane Vesicles of Eschrichia coli: I. A Simple Method for Preparing the Cytoplasmic and Outer Membranes

  Ichiro YAMATO, Yasuhiro ANRAKU, Kazushige HIROSAWA
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  ABSTRACT: 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 b 1 , NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.6.1.3], 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.
• Article: Sodium ion and proline binding sites in the Na+/proline symport carrier of Escherichia coli

  Kentaro Hanada, Takashi Yoshida, Ichiro Yamato, Yasuhiro Anraku
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  ABSTRACT: Proline binding activity of the Escherichia coli Na+/proline symport carrier is inhibited by a sulfhydryl reagent, N-ethylmaleimide (NEM). Proline and its analogs protected the carrier against the NEM-inactivation a Na+ (or Li+)-dependent manner. Na+ alone, even in the absence of proline, partially protected it from the NEM-inactivation. Mutant proline carriers, CS281, CS344 and CS349, which have a serine residue in place of Cys-281, Cys-344 and Cys-349, respectively (Yamato, I. and Anraku, Y. (1988) J. Biol. Chem. 263, 16055–16057) were also analyzed for cation-dependent proline binding and NEM-sensitivity. Proline binding activities of CS281 and CS344 were almost completely resistant to NEM, whereas that of CS349 was not. Furthermore, the proline binding activity of CS344 was remarkably lower than those of the wild-type, CS281 and CS349 carriers. These results indicate that Cys-344, which is located in the putative eighth membrane-spanning domain in the carrier, is a cysteine residue functionally involved in the high-affinity binding for sodium ion and proline.
  Biochimica et Biophysica Acta (BBA) - Biomembranes.