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Y Kaziro

Publications and source records attributed to Y Kaziro.

At least 181 records · Page 10Linked to original sources
PubMed1978

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 1. Purification and some properties of the purified factors.

Polypeptide chain elongation factors have been purified from an extreme thermophile, Thermus thermophilus HB8. By chromatography on a DEAE-Sephadex column, the factors were separated into two peaks; peak I contained a complex of EF-Tu and EF-Ts, while peak II was composed of EF-Tu.gdp and EF-G. These factors were subsequently purified to homogeneous states and crystallized. The EF-Tu . EF-Ts complex could be resolved into EF-Tu and EF-Ts by chromatography on a Sephadex G-200 column in the presence of 8 M guanidine-HCl. The complex could be reconstituted from EF-Tu and the renatured EF-Ts. No immunological cross-reaction was detected between EF-Tu, EF-Ts, and EF-G from T. thermophilus and the antibodies to their corresponding Escherichia coli factors. The molecular weight of EF-Tu . GDP determined by sedimentation equilibrium and sodium dodecylsulfate/polyacrylamide gel electrophoresis was 49000 and 51000 respectively. On the other hand, the molecular weight of EF-Ts was estimated as 27000 and 64000, respectively, by sodium dodecylsulfate/polyacrylamide gel electrophoresis and Sephadex gel filtration, suggesting that the protein existed probably as a dimer. The molecular weight of the EF-Tu . EF-Ts complex determined by sedimentation equilibrium and by gel filtration, was 142000 and 220000, respectively. Since the molar ratio of EF-Tu to EF-Ts in the EF-Tu . EF-Ts complex was one to one, it was suggested that the complex was composed of 2 mol each of EF-Tu and EF-Ts. The molecular weight of EF-G was estimated as 85000, 80000 and 78000 by equilibrium centrifugation, gel filtration, and sodium dodecylsulfate/polyacrylamide gel electrophoresis respectively.

Crystallization↗
PubMed1978

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 2. Catalytic properties.

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli. 1. The formation of a ternary guanine-nucleotide . EF-Tu . EF-Ts complex was demonstrated by gel filtration of the T. thermophilus EF-Tu . EF-Ts complex on a Sephadex G-150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli. 2. The dissociation constants for the complexes of T. thermophilus EF-Tu . EF-Ts with GDP and GTP were 6.1 x 10(-7) M and 1.9 x 10(-6) M respectively. On the other hand, T. thermophilus EF-Tu interacted with GDP and GTP with dissociation constants of 1.1 x 10(-9) M and 5.8 x 10(-8) M respectively. This suggests that the association of EF-Ts with EF-Tu lowered the affinity of EF-Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction. 3. Although the T. thermophilus EF-Tu . EF-Ts complex hardly dissociates into EF-Tu and EF-Ts, a rapid exchange was observed between free EF-Ts and the EF-Tu . EF-Ts complex using 3H-labelled EF-Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides. 4. Based on the above findings, an improved reaction mechanism for the regeneration of EF-Tu . GTP from EF-Tu . GDP is proposed. 5. Studies on the functional interchangeability of EF-Tu and EF-Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination. 6. T. thermophilus EF-Ts could bind E. coli EF-Tu to form an EF-Tu (E. coli) . EF-Ts (T. thermophilus hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF-Ts. On the other hand, no stable complex between E. coli EF-Ts and T. thermophilus EF-Tu has been isolated. 7. The uncoupled GTPase activity of T. thermophilus EF-G was much lower than that of E. coli EF-G. T. thermophilus EF-G formed a relatively stable binary EF-G . GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF-G . GDP and EF-G . GTP were 6.7 x 10(-7) M and 1.2 x 10(-5) M respectively. The ternary T. thermophilus EF-G . GDP . ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF-G.

Escherichia coli↗
PubMed1978

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.

Amino Acids↗
PubMed1977

A possible involvement of cya gene in the synthesis of cyclic guanosine 3':5'-monophosphate in E. coli.

Based on the following genetical experiments, the cya gene in E. coli was shown to be involved in the synthesis of both cyclic AMP and cyclic GMP. First, all five independent cya-deficient mutants accumulated exceedingly low amounts of cyclic GMP. Second, the ability to form both cyclic AMP and cyclic GMP was simultaneously restored by transduction of an intact cya locus to one of the above cya-deficient mutants. Third, a spontaneous revertant from one of the above mutants regained the synthetic activity for cyclic GMP as well as for cyclic AMP. Fourth, the characteristic of a strain overproducing cyclic GMP was co-transduced with the cya locus. These results suggest that the synthesis of both cyclic GMP and cyclic AMP is mediated by the same enzyme, adenylate cyclase, Interestingly, a reciprocal effect of glucose starvation was observed on the accumulation of both cyclic nucleotides. The formation of cyclic AMP was greatly enhanced on glucose starvation, whereas that of cyclic GMP proceeded at a slower rate than in the presence of glucose. This effect was observed only in cells carrying normal cya and crp genes, but not in a cya-altered or a crp-deficient strain.

Adenylyl Cyclases↗
PubMed1977

Translocation reaction promoted by polypeptide chain elongation factor-2 from pig liver.

Translocation of peptidyl-tRNA from the ribosomal A- to the P-site in the eukaryotic system was extensively investigated using a model system, in which translocation of Phe-tRNA from the A- to the P-site was examined by using the puromycin reaction, and the following results were obtained. 1) The puromycin reaction but not the translocation reaction proceeded at 0 degrees C. Since the latter could be demonstrated at 30 degrees C, it was possible to analyze translocation per se separately from the puromycin reaction. 2) Translocation was completely dependent on the elongation factor-2 (EF-2) and required the presence of GTP, which could be replaced by GMP-P(NH)P provided that the stoichiometric amount of EF-2 with respect to the amount or ribosomes was present. It was further demonstrated that translocation observed in the presence of GTP was catalytic, while that in the presence of GMP-P(NH)P was stoichiometric, indicating that hydrolysis of GTP was required for the catalytic reutilization of EF-2. 3) Translocation promoted by EF-2 in the presence of GMP-P(NH)P could be reversed, which suggests that hydrolysis of GTP is indispensable of the translocation reaction to proceed catalytically and unidirectionally forward.

Animals↗
PubMed1977

Involvement of 30S ribosomal protein S1 in poly(U)-directed polyphenylalanine synthesis.

The effect of 30S ribosomal protein S1 on poly(U)-directed polyphenylalanine synthesis was studied using a highly purified cell-free system which was devoid of endogenous S1. The system consisted of homogeneous preparations of EF-Tu, EF-Ts, and EF-G, and 70S ribosomes from which protein S1 had been removed by poly(U)-cellulose column chromatography. It was found that protein S1 was indispensable for translation of poly(U) by an S1-depleted system at low concentrations of poly(U). On the other hand, at higher concentrations of poly(U), a considerable amount of polyphenylalanine was synthesized in the absence of added S1. The stimulatory effect of S1 was observed at all Mg2+ concentrations examined but was most pronounced at 10 mM Mg2+. Some physicochemical properties of the protein were also studied. It was demonstrated that the protein has an elongated shape with an axial ratio of approximately 8.5.

Escherichia coli↗
PubMed1977

Structural fluctuation of the polypeptide-chain elongation factor Tu. A comparison of factors from Escherichia coli and Thermus thermophilus HB8.

The kinetics of hydrogen-deuterium exhcange in the polypeptide chain elongation factor Tu (EF Tu) from Escherichia coli and that from Thermus thermophilus HB8 has been examined in aqueous solutions at various pH and temperatures by means of infrared absorption measurements. The free EF-Tu from E. Coli has a greater reaction rate at all pH values and at every temperature than that of the GTP-bound or GDP-bound EF-Tu. The free EF-Tu from T. thermophilus, on the other hand, has an alomst equal reaction rate to that of EF-Tu-GDP in the temperature range 38-55 degrees C. For the peptide NH groups belonging to a medium-labile kinetic class, a small but definite difference in the rate of exchange reaction was observed between EF-Tu-GDP and EF-Tu-GTP for both E. coli and T. thermophilus. For less labile peptide NH groups, on the other hand, the rate of the exchange reaction with EF-Tu-GDP from T. thermophilus is only slightly affected by the pH of the solution at 38 degrees C and 45 degrees C, while the rate constant(k) with E. coli EF-Tu-GDP is pH-dependent (log k oc pH). For T. thermophilus EF-Tu, heat stability measurements, kinetics of the rates of GDP and GTP dissociation, and circular dichroic measurements have also been made. The molecular basis for the thermostability of T. thermophilus EF-Tu is discussed.

Circular Dichroism↗
PubMed1977

Purification and properties of the polypeptide chain release factor (RF-4) from an extreme thermophile, Thermus thermophilus HB8.

The polypeptide chain release factor 1 (RF-1) has been purified from an extreme thermophile, Thermus thermophilus HB8. The purification procedure included steps of aqueous two-phase partition, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, Sephadex G-150, and CM-Sephadex. The preparation was more than 90% pure as judged by polyacrylamide gel electrophoresis. The specific activity was about 3.3 pmol of formyl-[3H]-methionine released in 1 min at 25 degrees C per microgram of protein under the standard assay conditions using 4 pmol of the initiation complex and 1 nmol of UpApG. The molecular weight as determined by gel filtration on Sephadex G-150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58,000 and 45,000, respectively. As expected, the factor was extremely heat-stable, 50% of its activity remaining after incubation for 5 min at 84 degrees C. Several properties of the reaction catalyzed by RF-1 are also described.

Bacterial Proteins↗
PubMed1976

Purification and properties of a new polypeptide chain elongation factor, EF-1beta, from pig liver.

Eukaryotic polypeptide elongation factor 1 (EF-1) from pig liver has been resolved into two complementary factors, EF-1alpha and EF-1beta (Iwasaki, K., Mizumoto, K., Tanka, M., and Kaziro, Y. (1973) J. Biochem. (Tokyo) 74, 849). This paper describes the procedures for purification of EF-1beta and some properties of the purified factor. The purification method includes an aqueous two-phase separation technique, a treatment of the crude factor with sodium cholate and two successive column chromatographies on diethyl-aminoethyl-Sephadex A-50. By this method, EF-1beta was purified about 50-fold starting from the material obtained after two-phase separation followed by ammonium sulfate fractionation with a recovery of 20%. The purified EF-1beta appeared homogeneous, having a molecular weight of about 90,000. It consisted of two unequal subunits of the molecular weights of 55,000 and 30,000. It stimulates polymerization of phenylalanine dependent on poly(U) in the presence of both EF-1alpha and EF-2, as well as the EF-1alpha-dependent binding of phenylalanyl-tRNA to ribosomes in the presence of GTP. However, it had no effect on the stoichiometric binding of phenylalanyl-tRNA to ribosomes dependent on EF-1alpha in the presence of guanyl-5'-yl methylenediphosphonate. These results indicate that the function of EF-1beta is to stimulate the recycling of EF-1alpha.

Animals↗