Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.
Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.
复制标题
人血浆α-半胱氨酸蛋白酶抑制剂。
作者：
A. Gounaris;M. Brown;A. Barrett
采用S-羧甲基木瓜蛋白酶-琼脂糖凝胶亲和层析和高分辨率阴离子交换层析两步法纯化人血浆α-半胱氨酸蛋白酶抑制剂（α CPI）。蛋白质以Mr约64 000的形式和Mr更高（约100 000）的材料获得。在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳与还原，这两种形式显示了一个主要组成部分的先生64 000。针对α CPI提出了抗血清，“火箭”免疫测定显示19人血清中的平均浓度为35.9 mg/dl。两个低先生和高先生形式的α CPI被证实是唾液酸糖蛋白的电泳迁移率降低后，神经氨酸酶处理。α CPI在免疫学上与抗凝血酶III和α 1-抗胰凝乳蛋白酶不同，这两种血浆丝氨酸蛋白酶抑制剂具有相似的Mr值。α CPI也不同于半胱氨酸蛋白酶抑制剂A和B，这两种来自人肝脏的细胞内低Mr半胱氨酸蛋白酶抑制剂。复合物的α CPI与木瓜蛋白酶的免疫电泳检测，但解离游离酶和完整的抑制剂在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳。木瓜蛋白酶的结合的化学计量接近1：1的低先生和高先生的形式。发现α CPI是木瓜蛋白酶和人组织蛋白酶H和L的紧密结合抑制剂（Ki分别为34 pM、1.1 nM和62 pM）。相比之下，组织蛋白酶B的抑制弱得多，Ki约为35 μ M。二肽基肽酶I也被弱抑制。用菠萝蛋白酶消化α CPI产生了Mr约22，000的抑制片段，并将其分离出来。
Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.