title: Purification and characterization of a cysteine protease from corms of freesia, Freesia reflacta.
abstract: A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M(r) was estimated to be about 26,000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P1 positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P2 position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.
authors.affiliation: Department of Chemistry, Faculty of Science, Kagoshima University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kaneda
authors.forename: H
authors.initials: H
authors.lastname: Yonezawa
authors.forename: T
authors.initials: T
authors.lastname: Uchikoba
mesh_terms: D000595:Amino Acid Sequence; D002364:Caseins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology*; D010944:Plants / Q000201:enzymology*; D013696:Temperature
chemical_list: D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D010455:Peptides; D003546:Cysteine Endopeptidases