caa a22 4500 397555997 CHVBK 20180308164802.0 cr unu---uuuuu 161202e199601 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021212 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021212 Purification and Characterization of Bleomycin Hydrolase, Which Represents a New Family of Cysteine Proteases, from Rat Skin [Elektronische Daten] [Atsushi Takeda, Dousei Higuchi, Takako Yamamoko, Yoshiko Nakamura, Yutaka Masuda, Takahiro Hirabayashi, Kazuyasu Nakaya] Bleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in the β-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercap-toethanol: 45-65% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S−200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical sub units. The amino acid sequence of its NH2 -terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the ami no acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km, and Kmax values were 2.1 mM and 6.8 μmol.mg−1.h−1 for PLM, and 1.8 mM and 7.2 μmol.mg−1.h−1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and P-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-β-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics. © by the Journal of Biochemistry Regular Papers nationallicence antibiotics nationallicence bleomycin nationallicence bleomycin hydrolase nationallicence cysteine protease nationallicence rat skin nationallicence Takeda Atsushi Departments of Clinical Pathology 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut Higuchi Dousei Departments of Dermatology, Showa University Fujigaoka Hospital 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut Yamamoko Takako Departments of Clinical Pathology 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut Nakamura Yoshiko Departments of Clinical Pathology 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut Masuda Yutaka Department of Biochemistry, School of Pharmaceutical Sciences, Showa University 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142 aut Hirabayashi Takahiro Departments of Dermatology, Showa University Fujigaoka Hospital 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut Nakaya Kazuyasu Department of Biochemistry, School of Pharmaceutical Sciences, Showa University 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142 aut The Journal of Biochemistry Oxford University Press 119/1(1996-01), 29-36 0021-924X 119:1<29 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021212 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021212 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Takeda Atsushi Departments of Clinical Pathology 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut NATIONALLICENCE 700 1- Higuchi Dousei Departments of Dermatology, Showa University Fujigaoka Hospital 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut NATIONALLICENCE 700 1- Yamamoko Takako Departments of Clinical Pathology 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut NATIONALLICENCE 700 1- Nakamura Yoshiko Departments of Clinical Pathology 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut NATIONALLICENCE 700 1- Masuda Yutaka Department of Biochemistry, School of Pharmaceutical Sciences, Showa University 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142 aut NATIONALLICENCE 700 1- Hirabayashi Takahiro Departments of Dermatology, Showa University Fujigaoka Hospital 1-30 Fujigaoka, Aoba-ku, Yokohama 227 aut NATIONALLICENCE 700 1- Nakaya Kazuyasu Department of Biochemistry, School of Pharmaceutical Sciences, Showa University 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/1(1996-01), 29-36 0021-924X 119:1<29 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford