A Novel Assay Method for Aminopeptidase P and Partial Purification of Two Types of the Enzyme in Escherichia coli(Biological Chemistry)
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概要
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A new assay for aminopeptidase P was established by coupling with proline iminopeptidase using Gly-Pro-chromogen (e.g. Gly-Pro-β-naphthylamide, Gly-Pro-p-nitroanilide, or, Gly-Pro-4-methylcoumarin amide) as the substrate. With each substrate, a liner relationship was established between the enzyme amounts and color development or fluorescence due to the chromogen released. This assay method did not suffer from interference by materials in culture broth. By using this assay method, aminopeptidase P was partially purified from Escherichia coli HB101 by chromatographies on DEAE-Sephadex and high performance liquid chromatography (HPLC). On the chromatogram with a DEAE-Sephadex column, two peaks of aminopeptidase P were observed and were named APP-I and APP-II. APP-I was further purified by HPLC using DEAE-5PW and Phenyl-5PW columns. Optimum pHs of APP-I and APP-II were 8.0 and 9.0, respectively. In contrast to APP-I which was stable around Ph 10, APP-II was stable at pH 8 to 9. After incubation for 30 min at pH 8.0, fifty percent of the remaining activity of APP-I and APP-II were observed at 60℃ and 50℃. APP-I and APP-II were activated 3-fold by the addition of 5 and 30μM Mn^<2+>. They were inhibited by EDTA, and reactivated by adding Mn^<2+>. The molecular weights of APP-I and APP-II were 350,000 and 210,000, respectively. Each enzymes released the amino terminal amino acid when proline is at the penultimate position. The velocity of hydrolysis by the enzymes was not significantly different for most X-Pro bonds (X=amino acid) of peptides except for Pro-Pro bond. APP-II hydrolyzed penta-(Pro-Pro-Gly) at a much higher rate than APP-I, suggesting the aminopeptidase P reported by Yaron and Mlynar (BBRC, 32, 658 (1968)) to be APP-II.
- 社団法人日本農芸化学会の論文
- 1988-08-23
著者
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YOSHIMOTO TADASHI
School of Pharmaceutical Sciences, Nagasaki University
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TSURU DAISUKE
School of Pharmaceutical Sciences, Nagasaki University
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Yoshimoto Tadashi
School Of Pharmaceutical Sciences Nagasaki University
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Yoshimoto Tadashi
School Of Pharmaceutical Sciences Nagasaki University:yakult Central Institute For Microbiological R
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Tsuru D
School Of Pharmaceutical Sciences Nagasaki University:yakult Central Institute For Microbiological R
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Tsuru Daisuke
Department Of Applied Microbial Technology Sojo University
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MURAYAMA Nobuhiro
School of Pharmaceutical Sciences, Nagasaki University
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Murayama Nobuhiro
School Of Pharmaceutical Sciences Nagasaki University
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Murayama Nobuhiro
School Of Pharm. Sci. Showa University
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Tsuru Daisuke
School Of Pharmaceutical Sciences Nagasaki University
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Yoshimoto Tadashi
School of Pharamaceutical Sciences, Nagasaki University
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