Kopitz J, Kisen G O, Gordon P B, Bohley P, Seglen P O
Physiological-Chemical Institute, University of Tübingen, Federal Republic of Germany.
J Cell Biol. 1990 Sep;111(3):941-53. doi: 10.1083/jcb.111.3.941.
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.
在分离的大鼠肝细胞中，发现七种半衰期各异的胞质酶（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）以相同速率（3.5%/小时）被自噬隔离。自噬通过在蛋白酶抑制剂亮肽素存在的情况下，测量电破碎细胞的可沉降细胞器（主要是溶酶体）中酶活性的积累来进行测定。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，也能使催化活性酶（在溶酶体前液泡中）积累，这表明在溶酶体蛋白水解之前没有失活步骤发生。此外，亮肽素保护的完整性表明，溶酶体半胱氨酸蛋白酶对于自噬吞噬的蛋白质的初始蛋白水解攻击是必不可少的。这些实验表明，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大量过程，并且必须调用非自噬机制来解释不同酶的周转情况。
