Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.
Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.
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DOI：
10.1083/jcb.111.3.941
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发表时间：
1990-09
期刊：
影响因子：
--
通讯作者：
Seglen PO
中科院分区：
文献类型：
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作者：
Kopitz J;Kisen GO;Gordon PB;Bohley P;Seglen PO
七种具有不同半衰期的胞质酶（鸟氨酸脱羧酶，0.9 小时；酪氨酸转氨酶，3.1 小时；色氨酸加氧酶，3.3 小时；丝氨酸脱水酶，10.3 小时；葡萄糖激酶，12.7 小时；乳酸脱氢酶，17.0 小时；醛缩酶，17.4 小时）被发现具有自噬性被隔离在 在离体大鼠肝细胞中具有相同的速率（3.5%/h）。自噬的测量是在蛋白酶抑制剂亮肽素存在下，电破坏细胞的可沉积细胞器（主要是溶酶体）中酶活性的积累。溶酶体融合过程的抑制剂（长春花碱和天冬酰胺）即使在没有蛋白水解抑制的情况下也允许催化活性酶（在前溶酶体液泡中）积累，这表明在溶酶体蛋白水解之前没有发生失活步骤。此外，亮肽素的完全保护表明，溶酶体半胱氨酸蛋白酶对于自噬蛋白的初始蛋白水解攻击是必需的。实验表明，自噬-溶酶体途径对正常胞质蛋白的隔离和降解是一个非选择性的整体过程，必须调用非自噬机制来解释酶周转的差异。
Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.