Chakraborti T, Ghosh S K, Michael J R, Chakraborti S
Department of Biochemistry and Biophysics, University of Kalyani, West Bengal, India.
Biochem J. 1996 Aug 1;317 ( Pt 3)(Pt 3):885-90. doi: 10.1042/bj3170885.
We have investigated the role of an aprotinin-sensitive protease in regulating Ca(2+)-ATPase activity and Ca2+ uptake (ATP-dependent and Na(+)-dependent) in microsomes of bovine pulmonary vascular smooth muscle during treatment with the O2(-.)-generating system hypoxanthine plus xanthine oxidase. Treatment of the smooth muscle microsomes with the O2(-.)-generating system produced a protease in a gelatin-containing zymogram with an apparent molecular mass of 16 kDa. This 16 kDa proteolytic protein was found to be inhibited by superoxide dismutase (SOD) and aprotinin but not by PMSF. Using polyclonal antiserum to aprotinin, we found that it is an ambient antiprotease of the smooth muscle microsomes. Treatment of the microsomes with the O2(-.)-generating system stimulated protease activity tested with a synthetic substrate N-benzoyl-DL-arginine p-nitroanilide and also enhanced Ca(2+)-ATPase activity. It also stimulated ATP-dependent Ca2+ uptake. In contrast, Na(+)-dependent Ca2+ uptake was found to be inhibited by the O2(-.)-generating system. Pretreatment of the microsomes with SOD and aprotinin preserved the increase in protease activity, Ca(2+)-ATPase activity and ATP-dependent Ca2+ uptake. In addition, O2(-.)-caused inhibition of the Na(+)-dependent Ca2+ uptake which was reversed by SOD and aprotinin. Pretreatment with PMSF did not cause any discernible alteration in the protease activity, Ca(2+)-ATPase activity. ATP-dependent Ca2+ uptake and Na(+)-dependent Ca2+ uptake in the microsomes caused by the O2(-.)-generating system. These results suggest that an aprotinin-sensitive protease plays a pivotal role in regulating Ca(2+)-ATPase and Ca(2+)-uptake activities in microsomes of pulmonary vascular smooth muscle under oxidant O2(-.)-triggered conditions.
我们研究了一种抑肽酶敏感蛋白酶在次黄嘌呤加黄嘌呤氧化酶产生超氧阴离子（O2(-.)）的系统处理牛肺血管平滑肌微粒体过程中，对Ca(2+)-ATP酶活性及Ca2+摄取（ATP依赖和Na(+)依赖）的调节作用。用产生O2(-.)的系统处理平滑肌微粒体，在含明胶的酶谱中产生一种表观分子量为16 kDa的蛋白酶。发现这种16 kDa的蛋白水解蛋白被超氧化物歧化酶（SOD）和抑肽酶抑制，但不被苯甲基磺酰氟（PMSF）抑制。使用抗抑肽酶的多克隆抗血清，我们发现它是平滑肌微粒体的一种内源性抗蛋白酶。用产生O2(-.)的系统处理微粒体，用合成底物N-苯甲酰-DL-精氨酸对硝基苯胺检测发现刺激了蛋白酶活性，同时也增强了Ca(2+)-ATP酶活性。它还刺激了ATP依赖的Ca2+摄取。相反，发现产生O2(-.)的系统抑制了Na(+)依赖的Ca2+摄取。用SOD和抑肽酶预处理微粒体可保留蛋白酶活性、Ca(2+)-ATP酶活性及ATP依赖的Ca2+摄取的增加。此外，O2(-.)引起的Na(+)依赖Ca2+摄取的抑制被SOD和抑肽酶逆转。用PMSF预处理未引起产生O2(-.)的系统导致的微粒体中蛋白酶活性、Ca(2+)-ATP酶活性、ATP依赖的Ca2+摄取及Na(+)依赖的Ca2+摄取有任何可察觉的改变。这些结果表明，在氧化剂O2(-.)触发的条件下，一种抑肽酶敏感蛋白酶在调节肺血管平滑肌微粒体中Ca(2+)-ATP酶和Ca(2+)-摄取活性方面起关键作用。
