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31.
Summary A cell-associated pullalanase (-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55 000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr-Ala-Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75–80° C and at 80–85° C, respectively. The enzyme was stable up to 90° C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae B. acidopullulyticus B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.Presented in part at the Annual Meeting of the Agricultural Chemical Society of Japan at Tokyo, April 2, 1987 (Abstracts, p 91)Offprint requests to: Y. Suzuki 相似文献
32.
Shinjiro Mitsuda Naoki Kobayashi Yoshiaki Matsuda Yasuharu Itagaki Akira Suzuki Eitaro Kumazawa Kanji Higashio Gosei Kawanishi 《Applied microbiology and biotechnology》1991,35(4):504-509
Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm3 per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed.
Offprint requests to: S. Mitsuda 相似文献
33.
Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF. 总被引:53,自引:15,他引:38
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Y Gotoh K Moriyama S Matsuda E Okumura T Kishimoto H Kawasaki K Suzuki I Yahara H Sakai E Nishida 《The EMBO journal》1991,10(9):2661-2668
MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization. 相似文献
34.
Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins. 总被引:29,自引:17,他引:12
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The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins. 相似文献
35.
A prospective study of the value of sputum cytology in the diagnosis of squamous cell carcinoma of the larynx and hypopharynx is reported. Sputum cytology established the diagnosis in 63.5% of the patients with laryngeal lesions and in 77.4% of the patients with hypopharyngeal lesions. In laryngeal cancer, a positive diagnosis by sputum cytology was related to clinical T factors (according to the TNM classification): while only 29.4% of T1 lesions were positively detected by sputum cytology, 63.3% of T2 lesions, 69.7% of T3 lesions and 79.2% of T4 lesions were so detected. In hypopharyngeal cancer, there was no discernible relationship between sputum cytodiagnosis and clinical T factors. Generally, there was only a small number of cancer cells present in the sputum in these cases. Some of the squamous cancer cells were not very conspicuous and would require careful screening of the sputum specimens to be detected. 相似文献
36.
Type B nucleoside-diphosphatase of rat brain. Purification and properties of an enzyme with high thiamin pyrophosphatase activity 总被引:1,自引:0,他引:1
Type B nucleoside-diphosphatase was purified from membranes of rat brain by solubilization with a non-ionic detergent and successive column chromatographies on DEAE-cellulose DE-52, concanavalin-A-Sepharose, Bio-Gel HT, blue-Sepharose CL-6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5'-phosphate inhibited thiamin-pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin-pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP-hydrolytic activity was 6.5 in the presence of Mg2+. 相似文献
37.
S Matsuda 《Journal of biochemistry》1988,104(6):989-990
Ca2+-binding of S-100 protein was studied using a Ca2+ electrode at pH 6.80. In the presence of 0.1 M KCl and 10 mM MgCl2 (ionic strength 0.13), Ca2+-binding to S-100 protein occurred in three steps with positive cooperativity. The numbers of bound Ca2+ ions in the three steps were 2, 2, and 4. The Ca2+-binding constants were 6.9 x 10(3) M-1, 2.9 x 10(3) M-1, and 3.7 x 10(2) M-1, respectively. The Ca2+-binding constants of the first and second steps obtained in the presence of 33.3 mM MgCl2 or 0.1 M KCl (ionic strength 0.10) were 1.4 times larger than those described above. This suggests that Mg2+ does not inhibit Ca2+-binding of S-100 protein. The increase of KCl concentration from 0.1 to 0.2 M caused a decrease of the Ca2+-binding constants to ca. 50%. 相似文献
38.
Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3 总被引:8,自引:0,他引:8
S Ohta M Yohda M Ishizuka H Hirata T Hamamoto Y Otawara-Hamamoto K Matsuda Y Kagawa 《Biochimica et biophysica acta》1988,933(1):141-155
The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps. 相似文献
39.
Y Wada T Matsuo I Katakuse T Suzuki T Azuma S Tsujino S Kishimoto H Matsuda A Hayashi 《Biochimica et biophysica acta》1986,873(2):316-319
A plasma prealbumin variant with a methionine-for-valine substitution at position 30 is closely associated with familial amyloidotic polyneuropathy (FAP) type I. Secondary ion mass spectrometry of the tryptic digest of a carrier's prealbumin could easily detect an abnormal peptide containing the substitution besides the normal peptide. This is a sensitive and reliable method for the diagnosis of FAP. 相似文献
40.
The effect of phospholipase C on two isozymes (alpha (+) and alpha forms) of rat brain (Na+ + K+)-ATPase and the temperature-dependence of their activities were investigated. Phospholipase C from Clostridium welchii inhibited the activities of the enzymes treated with and without pyrithiamin or N-ethylmaleimide, a preferential inhibitor of the alpha (+) form, but the extent of the inhibition was higher in the control enzyme than in the treated enzymes. The treatment of the (Na+ + K+)-ATPase with phospholipase C altered a ratio between high- and low-affinity components for ouabain inhibition. It also caused the similar change in a ratio between the alpha (+) and alpha forms of Na+-stimulated phosphorylation from [gamma-32P]ATP. These findings indicate that the alpha (+) form of rat brain (Na+ + K+)-ATPase is more sensitive to phospholipase C than the alpha form. Analysis of Arrhenius plots of the activities of the control and pyrithiamin-treated enzymes showed that there was a difference between the two enzymes in a break point. We suggest that two isozymes of rat brain (Na+ + K+)-ATPase differ in the interaction with phospholipids or in the lipid-environment. 相似文献