Biol. Chem. Hoppe-Seyler Vol. 373, pp. 459-464, July 1992 Characterization and Structure of Pineapple Stem Inhibitor of Cysteine Proteinases BRIGITA LENAR&£, ANKA RITONJA, BORIS TURK, IZTOK DOLENC AND Vrro TURK Department of Biochemistry, J. Stefan Institute, Jamova 39, 61000 Ljubljana, Slovenia. Summary The complete amino acid sequence of the inhibitor of cysteine proteinases from pineapple stem acetone powder was determined. The inhibitor consists of 52 amino acids and is composed of two polypeptide chains (41 and 11 amino acids) linked via disulphide bonds. It differs from already known sequences in one to four amino acids. Data from its amino acid sequence analysis clearly show that this inhibitor cannot be a member of the cystatin superfamily. The Kj values for papain, bromelain and cathepsin L were determined. Introduction Cysteine proteinase inhibitors are distributed in mammalian tissue and body fluids as well as in plants and insects. The most studied are cystatins, tight and reversible binding inhibitors of papain-like cysteine proteinases, which form a superfamily of sequentially homologous proteins [1]. The amino acid sequence of the recently discovered cathelin, a protein inhibitor of cathepsin L [2] and potato cysteine proteinase inhibitor [3] assigns that new families of cysteine proteinases may exist. About 15 years ago from pineapple stem acetone powder polypeptide isoinhibitors of Mr of about 5600 were isolated, partialy characterized and the primary structures of three of them were determined [4]. It was shown that this inhibitor typep is composed of longer (41 residues) and shorter (11 residues) peptide chains connected with disulphide bonds. Due to their complete structural difference with cystatins we isolated these inhibitors and characterized the fourth form of the inhibitor. Keywords: Cysteine proteinase inhibitor; stem bromelain inhibitor; primary structure. Enzymes: Bromelain (EC 3.4.22.4); Papain (EC 3.4.22.2); Cathepsin L (EC 3.4.22.15); Endopeptidase Gly-C Abbreviations: Bz, benzoyl; DTE, dithiothreitol; EDTA, ethylenediaminotetraacetic acid; HPLC, high performance liquid chromatography, Kj, the inhibition constant; MCA, (4-methyl-7-coumaryl)amide; NNap, 2-naphthylamide; Pyr, Pyroglutamic acid; Z, benzyloxycarbonyl. Copyright © by Walter de Gruyter & Co · Berlin · New York Brought to you by | Purdue University Libraries Authenticated Download Date | 5/24/15 5:13 AM