Proteolytic Measurement of Isoenzymes in Blood

利用纯化的胞质天冬氨酸转氨酶失活蛋白酶，研究了多种同工酶活性的蛋白水解测定方法，以及胞质天冬氨酸转氨酶选择性蛋白水解失活测定血清中线粒体天冬氨酸转氨酶的最佳条件。一些蛋白酶，如α -凝乳胰蛋白酶、枯草杆菌蛋白酶、蛋白酶401和蛋白酶K，也能特异性灭活胞内天冬氨酸转氨酶，但不能灭活线粒体天冬氨酸转氨酶。蛋白酶401和蛋白酶K在贮藏时热稳定性较好。蛋白酶401在6.0-10.0的pH范围内有效。在实验过程中，pH为9.5、白蛋白含量为0.5%的混合试剂被确定为细胞内天冬氨酸转氨酶失活和线粒体天冬氨酸转氨酶、乳酸脱氢酶和苹果酸脱氢酶保存的最佳条件。血清内源性蛋白抑制剂如α _1-抗胰蛋白酶和α _2-巨红蛋白对蛋白酶401和蛋白酶k无抑制作用，测定了总线粒体天冬氨酸转氨酶、载脂蛋白和全线粒体天冬氨酸转氨酶活性之和。胞质天冬氨酸转氨酶完全失活，载脂蛋白线粒体天冬氨酸转氨酶在5分钟内被吡哆醛5′-磷酸完全激活。该方法与免疫化学法的结果具有良好的相关性（r=0.994,n=145），回归曲线为Y=0.95X + 1.60 （X；蛋白水解法），在100 μ mol /L吡脲醛5′-磷酸存在下，该酶对全细胞和载脂蛋白天冬氨酸转氨酶的抑制活性最高可达5000 U/L，是上限的200倍以上，对线粒体载脂蛋白天冬氨酸转氨酶的激活最高可达2000 U/L。组内CVs较好（1.37 ~ 7.49%）。健康受试者血清总线粒体天冬氨酸转氨酶活性平均值分别为4.8 （SD 0.9）和1.8 (SD 0.8) U/L （n=154）。各种常见的干扰测试不影响这个分析。少

A proteolytic measurement of various isoenzymes activities and the optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase to determine mitochondrial aspartate aminotransferase in serum were investigated by use of purified bytosolic aspartate aminotransferase inactivating proteases. Some of the proteases, such as, alpha-chymotrypsin, subtilisin, protease 401 and proteinase K, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial aspartate aminotransferase. The protease 401 and proteinase K were the heat stable for storage. Protease 401 was found to be effective over a pH range 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of cytosolic aspartate aminotransferase and preservation of mitochondrial aspartate aminotransferase, lactic dehydrogenase, and malic dehydrogenase in the assay procedure. The presence of serum endogeneous protein inhibitors such … More as alpha_1-antitrypsin and alpha_2-macroglobin did not inhibit protease 401 and proteinase K.Total mitochondrial aspartate aminotransferase, the sum of apo- and holo-mitochondrial aspartate aminotransferase activity in human serum, was measured by using proposed method. Cytosolic aspartate aminotransferase is completely inactivated, and apo-mitochondrial aspartate aminotransferase is completely activated by pyridoxal 5' -phosphate within 5 min. Results by the proposed method correlated well with those by an immunochemical method (r=0.994,n=145) and the regression curve was Y=0.95X + 1.60 (X ; proteolytic method) and showed excellent inhibitory activity of the protease for holo-and apo-cytoslic aspartate aminotransferase up to 5000 U/L more than 200-fold the upper limit and activation of mitochondrial apo-aspartate aminotransferase up to 2000 U/L in the presence of 100 mumol of pyridoxal 5' -phosphate per liter. Within-run CVs were good (1.37-7.49%). Mean values for total mitochondrial aspartate aminotransferase activities in serum of the healthy subjects were 4.8 (SD 0.9) and 1.8 (SD 0.8) U/L, respectively (n=154). Various common interferents tested not affects this assay. Less