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Biomedical subjects

Y Kaziro

Publications and source records attributed to Y Kaziro.

At least 163 records · Page 9Linked to original sources
PubMed1980

Proton nuclear magnetic resonance study on the roles of histidine residues in the binding of polypeptide chain elongation factor Tu from Thermus thermophilus with aminoacyl transfer ribonucleic acid and guanine nucleotides.

Proton nuclear magnetic resonance (1H NMR) spectra were measured of the polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thermus thermophilus HB8 [Nakano, A., Miyazawa, T., Nakamura, S., & Kaziro, Y. (1979) Arch. Biochem. Biophys. 196, 233-238], in order to elucidate the environment around functionally important histidine residues. In the present study, the behavior of five histidine C2 proton signals was studied in more detail. A hydrogen-deuterium exchange experiment was carried out on the histidine C2 protons of free EF-Tu, and the previous assignments of C2 proton signals were revised in part. An analysis of the 1H NMR spectra of EF-Tu photooxidized under various conditions indicates that a histidine residue is located in the aminoacyl-tRNA binding site and is probably essential for the binding with aminoacyl-tRNA. A solvent-accessible histidine residue is found to lie near the aminoacyl-tRNA binding site. Furthermore, the effect of paramagnetic hexacyanochromate(III) ion on the 1H NMR spectra of free EF-Tu suggests that another histidine residue lies near the guanine nucleotide binding site.

Guanine Nucleotides↗
PubMed1980

The nucleotide sequence of the cloned tufA gene of Escherichia coli.

The 4 kb (8.5 % lambda units) EcoRI fragment harboring the tufA gene of Escherichia coli was cloned using plasmid pTUA1 (Shibuya et al., 1979) and its structure was analyzed. The nucleotide sequence of about 1500 base pairs, covering the C-terminal portion of elongation factor EF-G (fus gene), the intercistronic region between fus and tufA, the entire structural gene for tufA with the GUG initiation and UAA termination codons, and the 3' flanking region of tufA, was determined. Comparison of the tufA nucleotide sequence with the tufB sequence (An and Friesen, 1980) and the known amino acid sequence of EF-Tu (Arai et al., 1980) revealed that the products of genes tufA and tufB are identical except for one amino acid at the C-terminal, i.e., glycine for tufA and serine for tufB. Nucleotide differences between tufA and tufB were found at 13 positions. Among them, one in the initiation codon and the other one in the C-terminal amino acid codon had replacements at the first letter of the codons. The other eleven changes were in the third codon positions, which did not affect the amino acid coding. The pattern of codon usage in tufA and tufB is highly nonrandom, and remarkably similar to that in ribosomal protein genes, with the codons for the most abundant species of isoaccepting tRNAs being preferentially utilized (Post et al., 1979; Post and Nomura, 1980).

Bacterial Proteins↗
PubMed1980

Primary structure of elongation factor Tu from Escherichia coli.

The amino acid sequence of elongation factor Tu (EF-Tu) from Escherichia coli has been determined. EF-Tu is a single-chain polypeptide composed of 393 amino acids (Mr 43,225 for the species bearing COOH-terminal serine). The NH2-terminal serine is acetylated, and lysine-56 is partially methylated. The sites of facile tryptic cleavage are at arginines 44 and 58 and at lysine-263. The cysteinyl residues associated with aminoacyl-tRNA and guanosine nucleotide binding activities are residues 81 and 137, respectively. The COOH-terminal amino acid is heterogenous in that analyses of the COOH-terminal peptides isolated from different EF-Tu preparations gave position 393 as glycine and serine in ratios (Gly/Ser) ranging from about 0.7 to 3.

Amino Acid Sequence↗
PubMed1980

Selective modification of functionally distinct sulfhydryl groups of sarcoplasmic reticulum Ca2+,Mg2+-adenosine triphosphatase with N-ethylmaleimide.

Rabbit sarcoplasmic reticulum vesicles were treated with N-ethylmaleimide (NEM) at pH 7.0. At 1.5 mM NEM, only 4 SH groups per mol of ATPase peptide were modified in 25 min at 30 degrees C. Two of these are essential for Ca2+ transport, one being involved in E-P formation (SHF), and the other in its decomposition (SHD), whereas the other two are apparently non-essential (SHN and SHN'). SHN was modified first, followed by SHD, SHN', and SHF, in this order. Modification of SHD was accompanied by the loss of Ca2+-transport activity, while E-P forming activity survived until the least reactive one (SHF) was modified. At a lower NEM concentration (4 x 10(-5) M) SHN could be selectively modified without loss of the enzyme activity. SHF could be protected by adenyl-5'-yl-imidodiphosphate (AMP-P(NH)P) in the presence of Ca2+ ions, whereas SHD was not. SHD was distinctly less reactive in the absence of Ca2+ (less than 10(-7) M) than in its presence. Changes in the reactivity of these SH groups may be related to conformational changes of the ATPase molecule induced by the binding of Ca2+ and ATP.

Adenylyl Imidodiphosphate↗
PubMed1979

Cloning of an EcoRI fragment carrying E. coli tufA gene.

EcoRI fragments of the transducing phage lambda fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Apr) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and is EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA . DNA hybridization of RNA transcribed from phage lambda rifd18 carrying tufB with DNA from pTUAl.

Coliphages↗
PubMed1979

Studies on stringent control in a cell-free system. Regulation by guanosine-5'-diphosphate-3'-diphosphate of the synthesis of elongation factor Tu.

The biosynthesis of elongation factor Tu (EF-Tu) has been studied in a cell-free system with DNA of the transducing phage lambdarifd18 as a template. It was found that the synthesis of EF-Tu in this system was inhibited by about 60% in the presence of 0.3 to 0.6 mM guanosine-5'-diphosphate-3'-diphosphate (ppGpp). The syntheses of several ribosomal proteins encoded in this template, i.e. L1, L10, L11, and L7/L12, were also depressed, whereas those of phage lambda proteins were rather enhanced by the addition of ppGpp. By separating the reaction into two steps, i.e., transcription and translation, the effect of ppGpp was shown to occur at the level of transcription. Several analogs, such as guanosine-5'-diphosphate-3'-monophosphate (ppGp) and guanosine-5'-diphosphate (ppG), were without effect. The formation of mRNA for EF-Tu was assessed directly by specific hybridization with pTUA1 DNA carrying tufA gene. The results clearly indicated that the synthesis of tufB . MRNA was severely and selectively inhibited by ppGpp.

Bacteriophage lambda↗
PubMed1979

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.

Adenosine Triphosphatases↗
PubMed1979

Studies on 30S ribosomal protein S1 from E. coli. I. Purification and physicochemical properties.

1. The distribution of ribosomal protein S1 in subcellular fractions of E. coli was determined by radioimmunoassay. It was found that about 70%, 20% and 10% of protein S1 were present in the high salt (1.0 M NH4Cl)-washed ribosomes, the ribosomal wash and the S100 fraction, respectively. 2. Protein S1 was purified from unwashed ribosomes by an improved procedure which included: (i) extraction of protein S1 from unwashed ribosomes with 1.2 M LiCl and 1.0 M NH4Cl, (ii) ammonium sulfate fractionation, (iii) two successive column chromatographies on DEAE-Sephadex, and (iv) hydroxylapatite column chromatography. Purified protein S1 was homogeneous in polyacrylamide gel electrophoresis under native and denatured conditions. 3. The molecular weights determined by sedimentation equilibrium and by SDS-polyacrylamide gel electrophoresis were 83,000 and 70,000 respectively. The sedimentation coefficient was estimated as 3.0S by glycerol gradient centrifugation. The stokes radius determined by Sephadex G-200 gel filtration was 45 A. From these data, the frictional ratio of protein S1 was calculated to be 1.65, assuming the molecular weight and partial specific volume to be 70,000 and 0.736, respectively. Protein S1 had an elongated shape with an axial ratio of approximately 8.5. 4. Protein S1 contained 2 residues of half-cystine and about 10 residues of tryptophan. From CD measurements, the contents of alpha-helix and beta-structure were estimated to be 32 and 27%, respectively. 5. As reported by Kolb et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 2379-2383), and Draper et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 4786-4790), the intrinsic fluorescence of protein S1 was markedly quenched on interaction with poly(U). The maximal quenching was observed when 30 mol of poly(U) (as UMP residues) was added to one mol of the protein.

Amino Acids↗
PubMed1978

Coordination of levels of elongation factors Tu, Ts, and G, and ribosomal protein SI in Escherichia coli.

The amounts of the polypeptide chain elongation factors Tu, Ts, and G, and ribosomal protein SI were assessed under various growth conditions using three independent procedures: (a) Immunoprecipitation and gel electrophoresis, (b) radioimmune assay, and (c) activity measurements. It was demonstrated that, during balanced growth of E. coli, the intracellular levels of these proteins increased in proportion to the growth rate, and the ratio of EF-Tu:EF-Ts:EF-G:protein SI was 4-5:1:1:1, at all growth rates. The effects of isoleucine starvation on the rates of synthesis of these proteins were examined using a pair of isogenic stringent and relaxed strains. The syntheses of all these proteins were found to be under the influence of stringent control. These results indicate that in E. coli the syntheses of the above four proteins are regulated in a coordinated manner and are subject to stringent control.

Escherichia coli↗
PubMed1978

A new extragenic suppressor of cya mutation. Mutant cyclic AMP receptor protein with an increased affinity for cyclic AMP.

A strain bearing an extragenic suppressor of cya mutation was isolated as a second-site revertant of an adenylate cyclase deficient strain. The mutant was unable to synthesize cAMP but showed normal fermentation profiles and growth properties on a variety of carbon sources. The site of reversion was mapped in, or near, the structural gene for the cAMP receptor protein. Structural alteration of the protein was directly demonstrated by the following biochemical observations: (i) A 10-fold decrease in the dissociation constant for cAMP, (ii) an acidic shift in the isoelectric point, and (iii) the altered binding properties to lambdah80dlac ps DNA.

Carbohydrate Metabolism↗