Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture.
Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture.
复制标题
亮肽素和胃酶抑素对原代培养的成年大鼠肝细胞蛋白质周转的影响。
DOI：
10.1016/0003-9861(81)90152-1
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发表时间：
1981
影响因子：
3.9
通讯作者：
A. Ichihara
中科院分区：
文献类型：
--
作者：
K. Tanaka;N. Ikegaki;A. Ichihara
在培养2天的大鼠肝细胞中加入酸性蛋白酶抑制剂胃酶抑素（pepstatin）可迅速抑制其水解血红蛋白（Hb）的活性，但对水解α-N-苯甲酰基-dl-精氨酸-β-萘酰胺（BANA）的活性无影响。另一方面，加入亮抑酶肽（一种巯基蛋白酶的抑制剂）抑制BANA水解酶的活性，并在1天内使Hb水解酶的活性增加6倍。两种蛋白酶抑制剂均不影响蛋白质合成速率。用茚三酮法测定氨基酸从肝细胞向Hanks盐溶液中的释放。胃蛋白酶抑制素在2天内仅抑制释放15%，而亮抑酶素在10小时内抑制释放65%。这两种抑制剂对蛋白质的释放具有相加的抑制作用，表明它们抑制了不同基团蛋白质的降解。亮抑酶肽的抑制作用在10小时后逐渐降低，这与观察到的上述蛋白酶活性的诱导一致。对预标记30 h的肝细胞释放[14 C]亮氨酸的研究表明，亮抑肽酶敏感性蛋白酶优先参与半衰期较长的蛋白质的降解。另一方面，两种抑制剂对标记仅1 h的肝细胞释放[14 C]亮氨酸的影响相似。它们的抑制作用再次是相加的，但亮肽素在延长孵育时间后的抑制作用没有减少，这表明半衰期短的蛋白质不是诱导蛋白酶的底物。这些结果表明，在肝细胞中，具有较长半衰期的蛋白质被组织蛋白酶B降解的程度大于被组织蛋白酶D降解的程度，而具有较短半衰期的蛋白质被这两种蛋白酶降解的程度相同。
Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on [14C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of [14C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.