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81.
Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP). 相似文献
82.
An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a Km of 0.74 mM and a kcat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The Km and kcat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively. 相似文献
83.
The effect of alterations to the insulin receptor on the insulin sensitivity of isolated adipocytes was studied. Receptor changes were induced by treatment of adipocytes with either phospholipase C or trypsin. After enzyme treatment, binding of insulin to insulin receptors and insulin-mediated glucose metabolism were examined. Exposure of adipocytes to phospholipase C (2 units/ml) significantly increased insulin binding to the cells, but destroyed the ability of the cells to oxidize glucose. After treatment with trypsin (500 micrograms/ml) for 5 min, insulin binding to the adipocytes was significantly increased. This was shown to be due to an increase in insulin-receptor affinity. Metabolic studies showed that trypsin treatment led to an increase in basal glucose transport but markedly decreased the response to insulin at all concentrations tested. Adipocytes treated with trypsin showed no significant difference in basal glucose oxidation rates when compared with controls, but were less sensitive to insulin at low insulin concentrations, and showed a decreased maximum response at high insulin concentrations. In conclusion, these findings indicate a dissociation between induced changes in binding of insulin to insulin receptors and subsequent hormone action. The importance of post-receptor events in the biological action of insulin is highlighted. 相似文献
84.
Y Nakamura Y Ohta S Itoh A Haratake Y Nakano A Umeda H Shima N Tomoda 《Acta cytologica》1992,36(4):559-562
Cytologic, light and electron microscopic, and immunohistochemical studies were conducted on a case of elastofibroma. Aspiration cytology showed a characteristic "braidlike" or "fern leaf-like" structure. Immunohistochemically the accumulate was shown to be elastin. Transmission electron microscopy indicated electron-dense, granular aggregates surrounded by microfilaments and collagen, while scanning electron microscopy revealed balls with a ball-of-yarn-like structure consisting of small fibrils, probably of elastin. These structures are unique to this disease and useful for diagnosis. 相似文献
85.
Triacsins A, B, C, and D are new inhibitors of long chain acyl-CoA synthetase (EC 6.2.1.3) and possess different inhibitory potencies against the enzyme (Tomoda, H., Igarashi, K., and Omura, S. (1987) Biochim. Biophys. Acta 921, 595-598). Acyl-CoA synthetase activity in the membrane fraction of Raji cells was also inhibited by triacsins. The same hierarchy of inhibitory potency as that against the enzyme from other sources, triacsin C greater than triacsin A much greater than triacsin D greater than or equal to triacsin B, was observed. When Raji cells were cultivated in the presence of triacsins, cell proliferation was inhibited in a dose-dependent fashion. The drug concentrations required for 50% inhibition of cell growth at day 2 were calculated to be 1.8 microM for triacsin A, much greater than 20 microM for triacsin B, 1.0 microM for triacsin C, and much greater than 15 microM for triacsin D, demonstrating a hierarchy for inhibitory potency of triacsins similar to that against the acyl-CoA synthetase activity. To understand the role of long chain acyl-CoA synthetase in animal cells, the effect of triacsins on the lipid metabolism of Raji cells was studied. When intact Raji cells were incubated with [14C]oleate in the presence of individual triacsins, the incorporation of [14C]oleate into each of the lipid fractions such as phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol was inhibited to an analogous extent. A common hierarchy, triacsin C greater than triacsin A much greater than triacsin D greater than triacsin B, was shown for the inhibition in each synthesis of the three lipids, which was identical with that for acyl-CoA synthetase. These findings indicate that the inhibition of acyl-CoA synthetase is well correlated with the inhibition of lipid synthesis. Taken together, the data strongly suggest that the inhibition of acyl-CoA synthetase by triacsins leads to the inhibition of lipid synthesis and eventually to the inhibition of proliferation of Raji cells. 相似文献
86.
Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems of various microorganisms and animal tissues. This antibiotic specifically blocks the activity of beta-ketoacyl thioester synthetase (condensing enzyme) by binding to the functional cysteine-SH in the active center of the condensing enzyme domain (the peripheral SH-group). However, fatty acid synthetase from C. caerulens is much less sensitive to cerulenin than fatty acid synthetases from other sources. The properties of C. caerulens synthetase were investigated and compared to those of Saccharomyces cerevisiae synthetase, which is sensitive to the antibiotic. The molecular weight of the enzymically active form of C. caerulens synthetase was 2.53 X 10(6). The enzyme consisted of two multifunctional proteins, alpha and beta, which are arranged in a complex, alpha 6 beta 6. The synthetase was inactivated by iodoacetamide. At 0 degrees C and pH 7.15, the second-order rate constant of k = 15.6 M-1 X s-1 was obtained for the inactivation by iodoacetamide. This value was about 15 times greater than that for S. cerevisiae synthetase. Treatment of C. caerulens synthetase with iodoacetamide, while impairing the synthetase activity, induced malonyl-CoA decarboxylase activity. When S. cerevisiae synthetase was preincubated with cerulenin, malonyl-CoA decarboxylase activity could not be detected even after treatment of the enzyme with iodoacetamide (Kawaguchi, A., Tomoda, H., Nozoe, S., Omura, S., & Okuda, S. (1982) J. Biochem. 92, 7-12). In the case of C. caerulens synthetase, on the other hand, malonyl-CoA decarboxylase activity was induced by iodoacetamide even after the preincubation of the enzyme with cerulenin.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
87.
H Tomoda A Kawaguchi T Yasuhara T Nakajima S Omura S Okuda 《Journal of biochemistry》1984,95(6):1712-1723
Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain. 相似文献
88.
T Matsunaga R Tomoda T Nakajima N Nakamura T Komine 《Applied and environmental microbiology》1988,54(6):1330-1333
We report a novel photochemical sterilization system in which Escherichia coli cells were sterilized with photosemiconductor powders (titanium oxide). For sterilization that could be used in practice, it was necessary to separate the TiO2 powders from the cell suspension. Therefore, semiconductor powders were immobilized on acetylcellulose membranes. We constructed a continuous-sterilization system consisting of a TiO2-immobilized acetylcellulose membrane reactor, a mercury lamp, and a masterflex pump. As a result, under the various sterilization conditions examined, E. coli (10(2) cells per ml) was sterilized to less than 1% survival when the cell suspension flowed in this system at a mean residence time of 16.0 min under irradiation (1,800 microeinsteins/m2 per s). We found that this system was reusable. 相似文献
89.
Pregnant Wistar rats were injected with bestatin, a specific inhibitor of aminopeptidase M. Placental aminopeptidase M activity was inhibited by injection of bestatin, and fetal body weight was statistically lower than that in the saline-injected or control group. Our present data suggest that placental aminopeptidase M plays an important role in fetal growth. 相似文献
90.
The absorption maxima in the Soret region and near 620nm of sulphhaemoglobin shifted from 419.5nm to 423nm and from 623nm to 619nm respectively with a decrease in oxygen concentrations of the sulphhaemoglobin solution [101.3, 20.3 and 0 kPa (760, 152 and 0 mmHg) partial pressures]. The major changes in the positions of the absorption maxima occurred drastically at oxygen concentrations between 20.3 kPa and 0 kPa, suggesting that the quaternary structure of sulphhaemoglobin has changed from the R to the T state. Inositol hexaphosphate, a known allosteric effector of haemoglobin, enhanced the shift in peak location. This result supports the view that the shift of the absorption maxima reflects changes in the conformation of sulphhaemoglobin. To investigate the relationship between the conformation of sulphhaemoglobin and the reactivity of the protein with various reagents, we studied the oxidation and reduction reactions of the protein with ascorbic acid, ferricyanide and nitrite under aerobic and anaerobic conditions. The results showed that the rates of oxidation and reduction of ferrous and ferric sulphhaemoglobins with these compounds are associated with the conformation of the protein. 相似文献