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131.
Analysis of the active center of Bacillus stearothermophilus neopullulanase. 总被引:5,自引:1,他引:4
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The active center of the neopullulanase from Bacillus stearothermophilus was analyzed by means of site-directed mutagenesis. The amino acid residues located in the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanse, Taka-amylase A, and other amylolytic enzymes. When amino acid residues Glu and Asp, corresponding to the putative catalytic sites, were replaced by the oppositely charged (His) or noncharged (Gln or Asn) amino acid residue, neopullulanase activities toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages disappeared. When the amino acids corresponding to the putative substrate-binding sites were replaced, the specificities of the mutated neopullulanases toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of neopullulanase participated in the dual activity toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. Pullulan is a linear glucan of maltotriosyl units linked through alpha-(1----6)-glucosidic linkages. The production ratio of panose from pullulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the alpha-(1----4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanse which exhibited higher specificity toward the alpha-(1----6)-glucosidic linkage. 相似文献
132.
Flavobacterium sp. strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, in which all of nylA (the gene for 6-aminohexanoate cyclic dimer hydrolase [EI]) was deleted but nylB (the gene for 6-aminohexanoate dimer hydrolase [EII]) was retained. KI725 showed no growth on unfractionated nylon oligomers (Nom1) obtained from a nylon factory as a sole carbon and nitrogen source (Nom1 minimum plate). Extracts of KI725 cells possessed hydrolytic activity for Nom1 (approximately 5% of the activity of KI72), but pOAD2-cured strains (KI722 and KI723) showed no activity. KI725R strains which grew on the Nom1 minimum plate were spontaneously isolated from KI725 at a frequency of 10(-7) per cell. Activity toward Nom1 was enhanced in KI725R strains (10 to 30% of the activity of KI72). This new Nom1 degrading enzyme (EIII, the nylC gene product) hydrolyzed not only Nom1 but also the N-carbobenzoxy-6-aminohexanoate trimer, a substrate which was not hydrolyzed by either EI or EII. Cloning and sequence analysis showed that the nylC gene is located close to nylB on pOAD21 and is a 1,065-bp open reading frame corresponding to 355 amino acid residues. The nucleotide sequence of the nylC gene and the deduced amino acid sequence of EIII had no detectable homology with the sequences of nylA (EI) and nylB (EII). 相似文献
133.
N Okada M Kobayashi H Ohtsuka S Shigematsu Y Nagasawa M Komatsu T Aizawa T Yamada 《Hormones et métabolisme》1992,24(9):429-433
Serum C-peptide immunoreactivity (CPR)/immunoreactive insulin (IRI) molar ratio was determined in 136 subjects without renal, hepatic and thyroid disorders, at fasting, and during the initial period of 75 g-oral glucose tolerance test. The subjects were divided into 4 groups based on their body weight and age; Group A, young (< 55 years) and normal body weight (body mass index [BMI, kg/m2] < or = 25) subjects; Group B, young and overweight (BMI > 25) subjects; Group C, aged (> or = 55 years) and normal body weight (BMI < or = 25) subjects; Group D, aged and overweight subjects. Fasting CPR/IRI ratio and absolute CPR level negatively correlated in Groups B and D but not in A and C. After oral glucose load with elevation of insulin, CPR/IRI ratio invariably declined in all groups and significant negative correlation between CPR/IRI and CPR was found in Groups A, B and D but not in C. Slope of the regression lines obtained for correlation between CPR/IRI ratio and CPR were significantly steeper at fasting compared to the post-stimulation phase. CPR/IRI ratio is affected by hyperinsulinemia and oral glucose load but not by obesity alone. Assuming that CPR/IRI ratio reflects hepatic extraction of insulin, the insulin clearance at fasting is progressively reduced with increasing insulin secretion in overweight subjects: failure to detect such phenomenon in normal body weight subjects may be due to a narrower CPR range in this population. Insulin metabolism at fasting and during glucose stimulation is likely to be regulated by distinct factors. 相似文献
134.
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
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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
135.
Masanao Watanabe Keiji Itoh Kuniya Abe Toshifumi Akizawa Kohji Ikenishi Mitsuru Furusawa 《Development, growth & differentiation》1992,34(2):223-231
In order to investigate whether a vasa -like protein is present in germ line cells of Xenopus , antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein.
Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 103 in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 103 band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila . 相似文献
Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 10
136.
GM1-gangliosidosis (genetic beta-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients 总被引:4,自引:2,他引:2
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GM1-gangliosidosis is a genetic neurological disorder caused by mutations in the lysosomal acid beta-galactosidase gene. While its phenotypic expression is complex, it is usually classified as being of infantile, juvenile, or adult form, on the basis of age at onset, the rate of symptomatic progression, and severity of central nervous system involvement. We have analyzed the acid beta-galactosidase gene in 12 Japanese patients from nine families. The aim was to identify mutations in individual patients and then to examine possible correlation between the mutations and the clinical phenotypes. Northern blotting studies with a full-length human beta-galactosidase cDNA showed that the mRNA ranged from undetectable to substantially decreased in the infantile patients but was normal in quantity and size in all juvenile and adult patients. Four distinct missense mutations have been identified, each limited to the respective clinical forms within our small-size samples. In the infantile patient with decreased but detectable mRNA, a point mutation was found resulting in Arg49----Cys. In the infantile patient with nearly undetectable mRNA, mutation Arg457----Ter was identified. The mutation Arg201----Cys was found in all four of the juvenile patients, while all six adult patients were homozygous for the point mutation Ile51----Thr. The mutations found in the juvenile and adult patients alter restriction sites in the normal gene and thus are amendable to quick screening. The prediction that these mutations are responsible for the clinical disease was confirmed by no expression of the catalytic activity of the mutant proteins in the COS-I cell expression system.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
137.
Y Shichida H Kandori T Okada T Yoshizawa N Nakashima K Yoshihara 《Biochemistry》1991,30(24):5918-5926
The photochemical and subsequent thermal reactions of 7-cis-rhodopsin prepared from cattle opsin and 7-cis-retinal were investigated by low-temperature spectrophotometry and laser photolysis, and compared with those of 11-cis-rhodopsin prepared from cattle opsin and 11-cis-retinal. Low-temperature experiments revealed that the absorption maxima of batho and lumi intermediates from 7-cis-rhodopsin were at slightly shorter wavelengths than those of 11-cis-rhodopsin while the meta I intermediates of both rhodopsin isomers showed the same absorption maxima. Kinetic experiments of the photobleaching process of 7-cis-rhodopsin using picosecond and nanosecond laser pulses revealed the formation of intermediates corresponding to the batho, lumi, meta I, and meta II intermediates from 11-cis-rhodopsin. An intermediate of 7-cis-rhodopsin corresponding to photorhodopsin (a precursor of bathorhodopsin), however, was not detected. Batho and lumi intermediates from 7-cis-rhodopsin had shorter lifetimes (approximately 40 ns and 300 microseconds) than those of 11-cis-rhodopsin (250 ns and 800 microseconds), but the lifetime of the meta I intermediate from 7-cis-rhodopsin was identical with that from 11-cis-rhodopsin (12 ms). These results indicate that the difference in configuration of the original chromophore between 7-cis- and 11-cis-rhodopsins is a cause of different chromophore-opsin interactions in the batho and lumi stages, while in the meta I stage the difference has disappeared by the relaxation of the protein near the chromophores. A possible interaction change between the 9-methyl group of the chromophore and its neighboring protein during the lumi-meta I transition will be discussed. 相似文献
138.
Nobuyuki Inagaki Hiroaki Nishimura Minoru Okada Hiroshi Mitsuhashi 《Plant cell reports》1991,9(9):484-487
Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium. 相似文献
139.
140.
Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment 总被引:7,自引:0,他引:7
Three X-linked mutations of Drosophila melanogaster, gs(1)N26, gs(1)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos. 相似文献