Murakami K, Etlinger J D
Proc Natl Acad Sci U S A. 1986 Oct;83(20):7588-92. doi: 10.1073/pnas.83.20.7588.
An endogenous inhibitor of high molecular weight protease was purified from human erythrocytes and partially characterized. The inhibitor was isolated by DEAE-Sephacel ion-exchange chromatography followed by separation on a Bio-Gel A-0.5m column. The inhibitor displayed a native Mr of 240,000 and contained a single subunit of Mr 40,000 after NaDodSO4/polyacrylamide gel electrophoresis. The Mr 240,000 hexamer inhibited high molecular weight protease noncompetitively (Ki = 8.3 X 10(-8) M) and showed marked susceptibility to proteolytic digestion and heat treatment. The purified factor was also a potent inhibitor of calcium-dependent protease (Ki = 2.8 X 10(-8) M), whereas it had no effect on trypsin, chymotrypsin, or papain. Heat treatment (50-70 degrees C X 10 min) caused loss of inhibition against high molecular weight protease; however, inhibition of calcium-dependent protease was stable under the same conditions. This result is consistent with different domains on the inhibitor that interact with high molecular weight protease and calcium-dependent protease. Together with earlier studies in which repression of inhibitor by an ATP-ubiquitin-dependent process was proposed, the present results suggest a general mechanism for regulation of multiple nonlysosomal proteases that are complexed with endogenous inhibitors.
从人红细胞中纯化出一种高分子量蛋白酶的内源性抑制剂，并对其进行了部分特性鉴定。通过DEAE-葡聚糖凝胶离子交换色谱法分离该抑制剂，随后在Bio-Gel A-0.5m柱上进行分离。该抑制剂的天然分子量为240,000，在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳后含有一个分子量为40,000的单一亚基。分子量为240,000的六聚体对高分子量蛋白酶具有非竞争性抑制作用（Ki = 8.3×10⁻⁸ M），并且对蛋白水解消化和热处理表现出明显的敏感性。纯化的因子也是钙依赖性蛋白酶的有效抑制剂（Ki = 2.8×10⁻⁸ M），而对胰蛋白酶、胰凝乳蛋白酶或木瓜蛋白酶没有作用。热处理（50 - 70℃×10分钟）导致对高分子量蛋白酶的抑制作用丧失；然而，在相同条件下对钙依赖性蛋白酶的抑制作用是稳定的。这一结果与抑制剂上与高分子量蛋白酶和钙依赖性蛋白酶相互作用的不同结构域一致。结合早期提出的通过ATP-泛素依赖性过程抑制抑制剂的研究，目前的结果提示了一种调节与内源性抑制剂复合的多种非溶酶体蛋白酶的一般机制。
