Hatanaka M, Yoshimura N, Murakami T, Kannagi R, Murachi T
Biochemistry. 1984 Jul 3;23(14):3272-6. doi: 10.1021/bi00309a023.
Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.
需要低钙和高钙的钙依赖性半胱氨酸蛋白酶形式分别被称为钙蛋白酶I和钙蛋白酶II。我们首次获得了针对钙蛋白酶I和钙蛋白酶II的单特异性抗体。使用这些抗体和电泳印迹法，我们发现，虽然大部分蛋白酶存在于细胞溶质中，但有少量可重复检测到的钙蛋白酶I与人类红细胞膜相关联。大多数与膜相关的钙蛋白酶I可用1%的 Triton X - 100提取，但不能用0.1%的去污剂提取。在存在0.1 mM钙离子和5 mM半胱氨酸的情况下，与膜相关的钙蛋白酶I优先降解膜蛋白带4.1，而对带3蛋白的降解则很缓慢。膜制剂的钙离子诱导的自消化被亮抑酶肽抑制，但添加到孵育培养基中的胞质钙蛋白酶抑制剂钙蛋白酶抑制素则没有这种作用。当在与检测钙蛋白酶I相同的条件下使用抗钙蛋白酶II抗体时，在红细胞的细胞溶质或膜中均未检测到钙蛋白酶II。
