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Biomedical subjects

H Kanazawa

Publications and source records attributed to H Kanazawa.

At least 325 records · Page 18Linked to original sources
PubMed1980

Organization of unc gene cluster of Escherichia coli coding for proton-translocating ATPase of oxidative phosphorylation.

The proton-translocating ATPase (F1-F0) of oxidative phosphorylation (ATP phosphohydrolase, EC 3.6.1.3) is coded for by a set of structural genes comprising the unc operon in Escherichia coli. We have analyzed several new transducing phages and plasmids carrying various lengths of the DNA segments of the unc operon by complementation assay using 14 new unc- mutants and representatives of previously described strains which were made available to us. Transducing phages carrying parts of the unc gene cluster were isolated: lambda uncA-9 and lambda glmS phages converted only some of the unc- mutants to the Unc+, as determined by complementation assays. A new hybrid plasmid (pMCR533) carrying part of the unc operon was constructed by inserting the HindIII fragment of lambda asn-5 DNA (a phage carrying the entire unc operon) into the unique HindIII site of pBR322. This plasmid transformed eight unc- strains to Unc+, including uncB402 and uncA401, but did not complement uncD11 or four other strains. Two minichromosomes which carry the E. coli replication origin were also tested: plasmid pNH05 transformed the uncB402 but not the uncA401 strain to Unc+, whereas plasmid pMCF1 transformed none of the mutants tested. Analysis of the DNAs from these transducing phages and plasmids with restriction endonucleases suggested that all of the structural genes for the F1-F0 complex are localized within a DNA segment of approximately 4.5 megadaltons containing two EcoRI sites. The approximate locations of the unc- mutations were mapped on this DNA segment.

Adenosine Triphosphatases↗
PubMed1980

Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli.

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.

Adenosine Triphosphatases↗
PubMed1980

Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.

Amino Acids↗
PubMed1979

Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn.

Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC. The transducing phages had been isolated from a strain of E. coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster [Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1978) Proc. Natl. Acad. Sci. USA 75, 5099--5103]. When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells. In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC. The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC. In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria. These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides. Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes.

Adenosine Triphosphatases↗
PubMed1979

Lipoprotein synthesis in Escherichia coli spheroplasts: accumulation of lipoprotein in cytoplasmic membrane.

Synthesis of cell envelope proteins was studied in ethylenediaminetetraacetic acid-lysozyme spheroplasts of Escherichia coli ML30. The rate of incorporation of [3H]arginine into proteins in spheroplasts was about 30% of that of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins synthesized in spheroplasts revealed the preferential synthesis of five polypeptides, one of which has been identified as the free form of murein lipoprotein. Lipoprotein synthesized in spheroplasts was found to be of same molecular size as that of mature lipoprotein. No prolipoprotein was observed even with a short pulse-labeling with [3H]arginine. On the other hand, significant accumulation of newly synthesized lipoprotein in the cytoplasmic membrane fraction of spheroplasts was observed. These results suggest that the processing of prolipoprotein occurs in the cytoplasmic membrane fraction of the cell envelope.

Bacterial Proteins↗
PubMed1978

An Escherichia coli mutant with an amino acid alteration within the signal sequence of outer membrane prolipoprotein.

Lipoprotein has been purified from an Escherichia coli strain carrying a mutation in the structural gene for murein lipoprotein (mlpA). Amino acid analysis of the purified mutant lipoprotein indicates that the mutant lipoprotein corresponds to the uncleaved prolipoprotein with a single amino acid replacement of glycine with aspartic acid. Automated Edman degradation has established the precise location of this amino acid substitution to be at the 14th residue of the prolipoprotein. This alteration in the signal sequence of prolipoprotein results in a failure of the mutated prolipoprotein to be processed. Furthermore, the structural alteration in the mutant lipoprotein appears also to have affected its topological localization in the mutant cell. Whereas lipoprotein in the wild-type strain is exclusively located in the outer membrane of the cell envelope, the membrane-bound lipoprotein in this mutant is recovered in both the inner and outer membranes of the cell envelope. The data suggest, however, that proteolytic cleavage of prolipoprotein to form mature lipoprotein is not essential for the translocation and assembly of lipoprotein into the outer membrane.

Amino Acid Sequence↗
PubMed1978

Acid proteases. II. Fluorescence study of the interaction of Cladosporium acid protease with glycyl-DL-norleucine methyl ester in the presence of cupric ions.

Glycyl-DL-norleucine methyl ester (GN), a diazoacetyl-DL-norleucine methyl ester (DAN) analog, in the presence of cupric ions was found to partially quench the protein fluorescence of acid protease from Cladosporium sp. No. 45-2, and cupric ions were also found to quench the fluorescence. These quenchings were pH-dependent. GN alone did not quench the fluorescence of the enzyme. The interaction between the enzyme and GN in the presence of cupric ions was studied statically at pH 5.4 in terms of fluorescence change. The dissociation constant, Kd, of the enzyme-GN complex in the presence of a 20-fold molar excess of cupric ions (0.08 mM) determined by fluorescence titration at 30 degrees C (Kd = 1.86 mM) was in good agreement with that obtained for GN from kinetics of inhibition of DAN-induced inactivation in the presence of a 20-fold molar excess of cupric ions at 30 degrees C (KA = 1.94 mM) (Kanazawa, H. (1977) J. Biochem. 81, 1739-1744). At various concentrations of cupric ions, no change of Kd was found. These results suggest that cupric ions are attracted to a negatively charged carboxyl group responsible for the formation of the enzyme-GN complex.

Binding Sites↗
PubMed1978

Transient repression of beta-galactosidase synthesis by glucose-6-phosphate in a mutant of Escherichia coli lacking enzyme II specific for glucose in the phosphoenolpyruvate-sugar phosphotransferase system.

The effects of glucose and glucose-6-phosphate in initiating the repression of beta-galactosidase synthesis were studied using a mutant of Escherichia coli K12 which lacks glucose-specific enzyme II of the phosphoenolpyruvate-sugar phosphotransferase system. It was found that glucose-6-phosphate causes transient repression of beta-galactosidase synthesis but glucose does not cause transient repression in this mutant. Evidence was obtained that both the presence of an active transport system for glucose-6-phosphate in the cells and glucose-6-phosphate in the medium are necessary for the initiation of transient repression. No metabolism of glucose-6-phosphate is required. Upon depletion of glucose-6-phosphate in the medium the transient repression was reversed. After the reversal the rate of enzyme synthesis was high in the cells which had been exposed to a high concentration of glucose-6-phosphate. It was concluded that the translocation of glucose-6-phosphate across the membranes is the primary event which affects both the initiation of and the recovery from the transient repression. During the transient repression the cellular content of cyclic adenosine 3',5'-monophosphate decreased significantly.

Biological Transport, Active↗
PubMed1978

Coupling factor ATPase from Escherichia coli. An uncA mutant (uncA401) with defective alpha subunit.

Inactive coupling factor ATPase (F1) was prepared from an uncoupled mutant (uncA401) of Escherichia coli. Reconstitution of ATPase activity was observed when alpha subunit from wild-type F1 was added to the dissociated inactive F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. ATPase was also reconstituted when the mixture of alpha subunit (wild type) and crude extract from the mutant was dialyzed against the same buffer. These results indicate that the mutant is defective in alpha subunit, suggesting that the uncA401 locus carries the structural gene for alpha subunit, and that this polypeptide plays an essential role in ATPase activity in F1 molecule.

Adenosine Triphosphatases↗