PYROGLUTAMYL PEPTIDASE FROM CHICKEN LIVER : PURIFICATION AND SOME PROPERTIES
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概要
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Pyroglutamyl peptidase (EC 3.4.19.3 : L-pyroglutamyl peptide hydrolase, pyrrolidonecarboxylyl peptidase) was purified from chicken liver by a series of column chromatographies on DEAE-Sephadex A-50,PCMB-T-Sepharose, Sephadex G-150 and hydroxyapatite. By these procedures, the enzyme was purified about 3800-fold with an activity recovery of 3.6%. The enzyme was most active and stable at pH7-8.2. Mercaptoethanol and ethylene diaminetetraacetate (EDTA) were found to stabilize the enzyme effectively. The enzyme was inactivated by the incubation with sodium tetrathionate, PCMB, Ni^<2+>, Cd^<2+>, Zn^<2+> and Hg^<2+>, but not affected by serine protease inhibitors such as diisopropylphosphoro fluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). Thiolprotease inhibitors, E-64 and Ep-475,were partially inhibitory to the enzyme. The molecular weight of the enzyme was estimated to be 86000 by the gel filtration method and the isoelectroc point was at pH 5.5 as checked by isoelectric focusing method. The enzyme readily hydrolyzed pyroglutamyl-β-naphthylamide (Pyr-2-NNap), Pyr-p-nitroanilide (Pyr-pNA), Pyr-4-methylcoumarinamide (Pyr-MCA) and most of Pyr-amino acids tested, by was only slightly active toward Pyr-D-Ala and completely inert toward Pyr-Pro. Kinetic analysis of the reaction indicated that the latter two peptides competitively inhibit the enzyme reaction with K_i values of 7.8 and 13.5 mM, respectively. The K_m values of Pyr-derivatives of 2-NNap, MCA, pNA and Ala were 0.7,0.04,0.73 and 0.89mM, respectively. The enzyme split bradykinin potentiator, neurotensin, luteinizing hormone releasing hormone (LHRH), thyroliberin (TRH), frog litorin and physalaemin, liberating pyroglutamic acid from their amino termini, but was inactive toward uperolein and TRH-analogue, piperidone carbonyl His-Pro-NH_2.
- 公益社団法人日本薬学会の論文
著者
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FUJIWARA Kunio
Faculty of Pharmaceutical Sciences, Nagasaki University
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Yoshimoto Tadashi
Faculty Of Pharmaceutical Sciences Nagasaki University
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Tsuru Daisuke
Faculty Of Pharmaceutical Sciences Nagasaki University
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Fujiwara Kunio
Faculty Of Pharmaceutical Sciences Nagasaki University
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SAKABE KAZUFUMI
Faculty of Pharmaceutical Sciences, Nagasaki University
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Sakabe Kazufumi
Faculty Of Pharmaceutical Sciences Nagasaki University
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