丝氨酸蛋白酶抑制剂α-1-蛋白酶抑制剂家族成员对大鼠肥大细胞蛋白酶I和II的差异性抑制作用。
Differential inhibition of rat mast cell proteinase I and II by members of the alpha-1-proteinase inhibitor family of serine proteinase inhibitors.
作者信息
Pirie-Shepherd S R, Miller H R, Ryle A
机构信息
Moredun Research Institute, Edinburgh, Scotland.
出版信息
J Biol Chem. 1991 Sep 15;266(26):17314-9.
Rat mast cell proteinase II (RMCP II) from mucosal mast cells was titrated into rat serum, and the resulting serine proteinase inhibitor (serpin)-enzyme complex was purified by affinity chromatography on anti-RMCP II-Sepharose 4B and by Mono-Q anion-exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCP II-Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) that inhibit RMCP II in rat serum. The kinetic constants characterizing the interaction between RMCP II and RSI and RSII are ka, 2.2 x 10(5) and 1.65 x 10(5) M-1 s-1, respectively; Ki, 3.6 x 10(-10) and 1.0 x 10(-9) M; and kd, 7.9 x 10(-5) and 1.65 x 10(-4) s-1. Amino-terminal sequence analysis indicated that RSI and RSII are distinct, differing at the amino-terminal residues, and are products of the rat Spi-1 locus. Rat mast cell proteinase I (RMCP I) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited.
将来自黏膜肥大细胞的大鼠肥大细胞蛋白酶II（RMCP II）滴定到大鼠血清中，然后通过抗RMCP II-琼脂糖4B亲和层析和Mono-Q阴离子交换对所得的丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂）-酶复合物进行纯化。纯化后的复合物用于制备多克隆抗体，该抗体在与RMCP II-琼脂糖4B交叉吸附后，对丝氨酸蛋白酶抑制剂具有特异性，并用于亲和纯化两种能抑制大鼠血清中RMCP II的大鼠丝氨酸蛋白酶抑制剂分子（RSI和RSII）。表征RMCP II与RSI和RSII之间相互作用的动力学常数分别为：结合常数（ka），分别为2.2×10⁵和1.65×10⁵ M⁻¹ s⁻¹；抑制常数（Ki），分别为3.6×10⁻¹⁰和1.0×10⁻⁹ M；解离常数（kd），分别为7.9×10⁻⁵和1.65×10⁻⁴ s⁻¹。氨基末端序列分析表明，RSI和RSII是不同的，在氨基末端残基处存在差异，并且是大鼠Spi-1基因座的产物。来自结缔组织肥大细胞的大鼠肥大细胞蛋白酶I（RMCP I）可切割RSI和RSII，且不受抑制。
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