Hirashiki I, Ogata F, Yoshida N, Makisumi S, Ito A
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
J Biochem. 1990 Oct;108(4):604-8. 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-modified 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 VVAG sequence in this fragment corresponds to the conserved sequence QVVAG 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.
多花紫藤的种子含有几种半胱氨酸蛋白酶抑制剂（胱抑素）。我们纯化并鉴定了其中一种抑制剂，命名为WCPI - 3。通过凝胶过滤和SDS - PAGE法分别测得WCPI - 3的分子量为17,500和15,700。其等电点为5.7。WCPI - 3与天然木瓜蛋白酶形成等摩尔复合物，解离常数估计为6.1 nM。通过凝胶过滤或非变性PAGE分析未观察到WCPI - 3与Cys25修饰的木瓜蛋白酶（如S - 羧甲基化或S - 氨甲酰甲基化木瓜蛋白酶）之间形成复合物。由无色杆菌蛋白酶（赖氨酰内肽酶）消化WCPI - 3得到的一个肽片段具有氨基酸序列VVAGVNYRFVLK。该片段中的VVAG序列对应于保守序列QVVAG，被认为是与半胱氨酸蛋白酶结合的区域之一。WCPI - 3氨基末端部分（34个残基）的氨基酸序列与水稻种子中的水稻胱抑素高度同源。
