Kunimatsu M, Tada T, Narita Y, Ozaki Y, Liu Z Q, Shearer T R, Sasaki M
Department of Biochemistry, Nagoya City University Medical School, Japan.
Cardiovasc Pathol. 1999 Jan-Feb;8(1):7-15. doi: 10.1016/s1054-8807(98)00018-0.
Tissue damage resulting from ischemia due to myocardial infarction is thought to be intensified by the proteolytic action of endogenous enzymes. Calpain (calcium dependent cysteine protease) is considered to be a highly likely candidate, since it is activated by calcium ion which increases in concentration under conditions of ischemia. We prepared a mono-specific antibody against the active site histidine stretch, Lys-Leu-Val-Lys-Gly-His-Ala-Tyr-Ser-Val, in the calpain 80 kDa large subunit. The specificity of the antibody was verified by its inhibitory effect on the caseinolytic activity of both mu- and m-calpains, western blotting analysis, and by absorption with the antigen peptide. The antibody was used to localize the intracellular distribution of activated calpains in infarcted regions of the human heart. The results showed that myocardial cells affected by ischemia were stained by the antibody, allowing damaged cells to be distinguished from cells of unaffected regions and that the immunostained regions were essentially the same regions as those identified by dense eosinophilic staining with hematoxylin and eosin. However, the staining pattern obtained with the antibody, was characteristic in denser staining at the cell periphery, whereas the damaged cells were stained homogeneously by hematoxylin and eosin. By the former method, results of staining indicated that the activation site of the calpain proenzyme was in the peri-plasma membrane, whereas by the latter method, diffusely distributed plasma proteins such as albumin and immunoglobulins were visualized as demonstrated in earlier reports.
心肌梗死导致的缺血所引起的组织损伤被认为会因内源性酶的蛋白水解作用而加剧。钙蛋白酶（钙依赖性半胱氨酸蛋白酶）被认为是一个很有可能的候选因素，因为它会被钙离子激活，而在缺血状态下钙离子浓度会升高。我们制备了一种针对钙蛋白酶80 kDa大亚基中活性位点组氨酸序列（Lys-Leu-Val-Lys-Gly-His-Ala-Tyr-Ser-Val）的单特异性抗体。通过其对μ-钙蛋白酶和m-钙蛋白酶的酪蛋白水解活性的抑制作用、蛋白质印迹分析以及与抗原肽的吸附作用，验证了该抗体的特异性。该抗体被用于定位人心脏梗死区域中活化钙蛋白酶的细胞内分布。结果显示，受缺血影响的心肌细胞被该抗体染色，从而能够将受损细胞与未受影响区域的细胞区分开来，并且免疫染色区域与苏木精和伊红染色显示的致密嗜酸性染色所确定的区域基本相同。然而，用该抗体获得的染色模式的特征是在细胞周边染色更密集，而受损细胞被苏木精和伊红均匀染色。通过前一种方法，染色结果表明钙蛋白酶原酶的激活位点位于质膜周围，而通过后一种方法，如早期报道所示，可观察到白蛋白和免疫球蛋白等弥漫分布的血浆蛋白。
