Purification and complex formation analysis of a cysteine proteinase inhibitor (Cystatin) from seeds of Wisteria floribunda

• Published in: Journal of biochemistry (Tokyo) Vol. 108; no. 4; pp. 604 - 608
• Main Authors: Hirashiki, I, Ogata, F, Yoshida, N, Makisumi, S, Ito, A
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.10.1990
• Subjects: Amino Acid Sequence; amino acid sequences; amino-terminal sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chickens; Chromatography, Gel; Cystatins - isolation & purification; Cystatins - pharmacology; cysteine proteinase; Cysteine Proteinase Inhibitors; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Hydrolysis; Isoelectric Focusing; isoelectric point; isolation; Molecular Sequence Data; Molecular Weight; Papain - isolation & purification; Plant Proteins - isolation & purification; Plant Proteins - pharmacology; proteinase inhibitors; purification; Rats; Seeds - analysis; Sequence Homology, Nucleic Acid; Serine Endopeptidases; Wisteria floribunda; Seeds; Purification; Gel electrophoresis; Cysteine proteinase; Enzyme inhibitor; Dissociation constant; Papain; Inhibitor enzyme complex; Binding site; Chromatography; Isoelectrical focusing; Leguminosae; Dicotyledones; Angiospermae; N terminal-Sequence; Gel filtration; Spermatophyta; Physicochemical properties
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123250

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modlfied papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The WAG sequence in this fragment corresponds to the conserved sequence QWAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.