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51.
O Nureki K Suzuki M Hara-Yokoyama T Kohno H Matsuzawa T Ohta T Shimizu K Morikawa T Miyazawa S Yokoyama 《European journal of biochemistry》1992,204(2):465-472
The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm. 相似文献
52.
Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins 总被引:21,自引:7,他引:14 下载免费PDF全文
N Imamoto Y Matsuoka T Kurihara K Kohno M Miyagi F Sakiyama Y Okada S Tsunasawa Y Yoneda 《The Journal of cell biology》1992,119(5):1047-1061
Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import. 相似文献
53.
K Sato T Akaike M Kohno M Ando H Maeda 《The Journal of biological chemistry》1992,267(35):25371-25377
To elaborate the catalytic activity of Cu2+ of Cu,Zn-superoxide dismutase (SOD) in the generation of hydroxyl radical (.OH) from H2O2, we investigated the mechanism of inactivation of alpha 1-protease inhibitor (alpha 1-PI), mediated by H2O2 and Cu,Zn-SOD. When alpha 1-PI was incubated with 500 units/ml Cu,Zn-SOD and 1.0 mM H2O2, 60% of anti-elastase activity of alpha 1-PI was lost within 90 min. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that free .OH was indeed generated in the reaction of Cu,Zn-SOD/H2O2; this was substantiated by the almost complete eradication of .OH by either ethanol or dimethyl sulfoxide accompanied by the generation of carbon-centered radicals. .OH production and alpha 1-PI inactivation in the H2O2/SOD system became apparent at 30 min or later. Dimethyl sulfoxide and 5,5-dimethyl-1-pyrroline N-oxide protected inactivation of alpha 1-PI significantly in this system, indicating that alpha 1-PI inactivation was mediated by .OH. SOD activity decreased rapidly during the reaction with H2O2 for the initial 30 min. Time-dependent changes in the ESR signal of SOD showed the destruction of ligands for Cu2+ in SOD by H2O2 within this initial period. Thus we conclude that inactivation of alpha 1-PI is mediated in the H2O2/Cu,Zn-SOD system via the generation of .OH by free Cu2+ released from oxidatively damaged SOD. 相似文献
54.
55.
Protocorms or protocorms with roots of an achlorophyllous orchidGaleola septentrionalis were inoculated with isolates ofRhizoctonia repens, R. solani, andRhizoctonia spp. The seedlings were infected with eight of twelve isolates ofR. repens. Fungal coils were formed in the cells, which was suggestive of a symbiotic association. The other isolates caused soft rot
or no infection to the protocorms or the protocorms with a root.
Contribution No. 97, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba,
Tsukuba, Ibaraki 305, Japan. 相似文献
56.
Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells 总被引:3,自引:0,他引:3
We have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of [32P]Pi-labeled and [3H]leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fractionation studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin. 相似文献
57.
58.
Keizo Kawamoto Mitsuhiko Akaboshi 《Origins of life and evolution of the biosphere》1982,12(2):133-141
The molecular basis for the chemical evolution of low molecular compounds was studied using electric discharges on a higly oxidized atmosphere comprised of CO2, N2 and H2O. In the gas phase, O2 and CO were formed by the decomposition of CO2 and their yields were enhanced by the addition of N2 to the gas mixture. It was demonstrated that H2O suppressed the reduction of CO2 while H2O also had a role in producing organic compounds such as formic acid and formaldehyde. Infrared analysis of the water soluble products and the inner surface of the reaction vessel indicated the production of compounds more complex than formic acid and formaldehyde. These compounds contained the chemical bonds which were identified to be OH, CO, CN and/or CC. 相似文献
59.
60.
NG, a strain of cultured tobacco cells of Nicotiana glutinosahad high growth rates and carboxylate contents (CA) of100 to 130 meq/100 g of dry cells on media containing 42 meqNO3/liter as the sole N source. (CA) is the amount ofinorganic cations minus inorganic anions in meq per 100 g ofdry cells. NG, cultured on media containing NH4 10+NO3 42 in meq/liter,had lower growth rates and lower (CA) values as comparedwith NG on media containing NO3 as the sole N source. NG, cultured on media containing NH4 30+NO3 42 in meq/liter,had high growth rates and (AC) values of 22 to 53 meq/100gof dry cells. In this case, the (AC) content may correspondto organic cations, basic organic N compounds such as free asprotein-bound basic amino acids. The easily absorbed Cl mayhave been required maintain good growth conditions such as ionicbalance and a favorable pH in the cells. Thus cultured cells of Nicotiana glutinosa may have physiologicaladaptability against variations in a relatively wide range of|CA| contents [|CA| being the absolute valuesof (CA)]. (Received May 15, 1980; ) 相似文献