Nakamura Y, Fukiage C, Ma H, Shih M, Azuma M, Shearer T R
Research Laboratories, Senju Pharmaceutical Co., Ltd., 1-5-4 Murotani, Nishi-ku, Kobe, 651-2241, Japan.
Exp Eye Res. 1999 Aug;69(2):155-62. doi: 10.1006/exer.1998.0686.
The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82-induced proteolysis in rodent lenses may occur even in the presence of calpastatin.
本研究的目的是在大鼠晶状体中测试三种钙蛋白酶抑制剂（重组钙蛋白酶抑制蛋白结构域I、E64和SJA6017）对Lp82钙蛋白酶的作用。Lp82是钙蛋白酶超家族中一种晶状体特异性同工酶，属于钙激活的半胱氨酸蛋白酶（EC 34.22.17）。通过酪蛋白酶谱法和免疫印迹法测定Lp82和m-钙蛋白酶的蛋白水解活性及蛋白质水平。还通过将大鼠晶状体可溶性和不溶性组分与钙进行体外孵育，测试内源性Lp82对波形蛋白的活性。大部分Lp82活性可被不可逆抑制剂E64和可逆抑制剂SJA6017抑制。然而，本研究的一个主要发现是，晶状体可溶性和不溶性组分中的Lp82对来自普遍存在的钙蛋白酶的内源性组织抑制剂钙蛋白酶抑制蛋白的重组结构域I的抑制作用，比m-钙蛋白酶更不敏感。通过使用重组钙蛋白酶抑制蛋白抑制内源性晶状体m-钙蛋白酶，我们能够证明Lp82的首个底物波形蛋白。这些数据表明，即使存在钙蛋白酶抑制蛋白，啮齿动物晶状体中由Lp82诱导的蛋白水解仍可能发生。
