Biol. Chem. Hoppe-Seyler Vol. 373, pp. 441-446, July 1992
Isolation and Characterization of Bovine Stefin B BORIS TURK, IGOR KRIZAJ AND Vrro TURK Dept. of Biochemistry, J. Stefan Institute, Jamova 39, 61000 Ljubljana, Slovenia
Summary A new cysteine proteinase inhibitor (CPI) was isolated from bovine thymus. According to the amino acid sequence it belongs to the stefin family. It appears as a monomer and a dimer with monomer Mr of 11,178 and pi values 5.6 for the monomer and 5.2 and 5.6 for the dimer. Kj for the interaction with papain was determined to be 0.12 nM. The most interesting feature of bovine stefin B is the replacement of the highly conserved QVVAG region in stefins with the QLVAG sequence without interfering its inhibitory properties. Introduction Stefin B is a tight and reversibly binding inhibitor of the cysteine proteinases [ 1 ]. It has been isolated from human [ 1-3] and rat [4]. The last one was originally named rat cystatin ß . The sequences of both inhibitors were determined [5,6] and human stefin B was cloned as well [7,8]. The mechanism of interaction of stefin B was proposed from the crystal structure of the human stefin B-papain complex [ 9 ]. Stefin B is a member of the stefin family which, together with the cystatin and the kininogen families, form the superfamily of the cystatins. The characteristics of the stefin family are: Mr 11,000, contains no carbohydrates and no intramolecular disulphide bonds, but has a highly conserved QWAG region in the first hairpin loop which interacts with the target enzyme [10-12]. However, it has already been shown that some exchanges within this region have only minor effect on the inhibitory properties of the stefins [13,14]. The objective of our study was to determine the presence of low-Mr inhibitors of the cysteine proteinases in bovine thymus, to isolate them in quantities sufficient for their characterization and to characterize them. Enzymes: Papain (EC 3.4.22.2). Abbreviations: CPI, cysteine proteinase inhibitor; Z, bezyloxycarbonyl; MCA, 7-(4-methyl)coumarylamide; Bz, benzoyl;
Isolation and Characterization of Bovine Stefin B BORIS TURK, IGOR KRIZAJ AND Vrro TURK Dept. of Biochemistry, J. Stefan Institute, Jamova 39, 61000 Ljubljana, Slovenia
Summary A new cysteine proteinase inhibitor (CPI) was isolated from bovine thymus. According to the amino acid sequence it belongs to the stefin family. It appears as a monomer and a dimer with monomer Mr of 11,178 and pi values 5.6 for the monomer and 5.2 and 5.6 for the dimer. Kj for the interaction with papain was determined to be 0.12 nM. The most interesting feature of bovine stefin B is the replacement of the highly conserved QVVAG region in stefins with the QLVAG sequence without interfering its inhibitory properties. Introduction Stefin B is a tight and reversibly binding inhibitor of the cysteine proteinases [ 1 ]. It has been isolated from human [ 1-3] and rat [4]. The last one was originally named rat cystatin ß . The sequences of both inhibitors were determined [5,6] and human stefin B was cloned as well [7,8]. The mechanism of interaction of stefin B was proposed from the crystal structure of the human stefin B-papain complex [ 9 ]. Stefin B is a member of the stefin family which, together with the cystatin and the kininogen families, form the superfamily of the cystatins. The characteristics of the stefin family are: Mr 11,000, contains no carbohydrates and no intramolecular disulphide bonds, but has a highly conserved QWAG region in the first hairpin loop which interacts with the target enzyme [10-12]. However, it has already been shown that some exchanges within this region have only minor effect on the inhibitory properties of the stefins [13,14]. The objective of our study was to determine the presence of low-Mr inhibitors of the cysteine proteinases in bovine thymus, to isolate them in quantities sufficient for their characterization and to characterize them. Enzymes: Papain (EC 3.4.22.2). Abbreviations: CPI, cysteine proteinase inhibitor; Z, bezyloxycarbonyl; MCA, 7-(4-methyl)coumarylamide; Bz, benzoyl;
