摘要
水稻的糠皮和胚经生理盐水浸取、离心后的上清液加热至８０℃处理１０ｍｉｎ，离心获得的上清液调ｐＨ至８．０，得到沉淀。沉淀溶解于０．０１ｍｏｌ／ＬＨＣｌ，经透析冷冻干燥得水稻巯基蛋白酶抑制剂（ＣＰＩ）粗品；粗品再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱线性离子梯度洗脱和ＳｅｐｈａｄｅｘＧ－１００柱分子筛层析，即可获得在ＰＡＧＥ、ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ上均为单一蛋白带的ＣＰＩ样品。经上述步骤，ＣＰＩ可被纯化５８倍。经ＳｅｐｈａｄｅｘＧ－１００和ＳＤＳ－ＰＡＧＥ测定其分子量均为１２０００，Ｎ末端氨基酸为Ｐｒｏ，等电点５．６．水稻ＣＰＩ经１００℃处理１０ｍｉｎ后，其抑制活性无任何变化，在ｐＨ２．０～９．０之间，活性也不发生改变，但ｐＨ在９．０以上，其活性逐渐下降，水稻ＣＰＩ对木瓜蛋白酶是一种高亲和性的抑制剂，它对木瓜蛋白酶和无花果蛋白酶有强抑制作用，对菠萝蛋白酶仅有弱抑制作用，但对胰蛋白酶则全无抑制作用；其抑制类型属竞争性抑制剂类型，Ｋ＿ｉ值约３．５×１０￣（－８）ｍｏｌ／Ｌ对木瓜蛋白酶的抑制摩尔比约为１：１。
A cysteine proteinase inhibitor(CPI) from seeds of rice (Xie Yiu-46) was purified by heating, adjusting PH, DEAE-Sepharose and Sephadex G-100 column. The CPI gave a single band on SDS-PAGE and HPLC with a molecular weight of 12 000 and a isoelectric point at PH5. 6. Nterminal amino acid was proline. The CPI was stable below 100℃ and between pH2.0-9.0. It can inhibite strongly papain and ficin, weakly to bromelin, but not to trypsin. The inhibition of papain by the CPI was competitive, with a K_i value of 3. 5×10￣(-8) mol/L and the mole ratio is about 1:1 .
出处
《生物化学杂志》
CAS
CSCD
1996年第6期703-708,共6页
基金
国家自然科学基金
关键词
水稻
巯基蛋白酶
抑制剂
提纯
性质
Cysteine proteinase inhibitor, proteinase inhibitor, Purification
