Katunuma N, Kominami E
Adv Enzyme Regul. 1985;23:159-68. doi: 10.1016/0065-2571(85)90045-7.
Cytoplasmic proteins are degraded with different half-lives in vivo. Large parts of proteins are believed to be degraded primarily in autophagic vacuoles-lysosomal system. However, the mechanism by which cell proteins are delivered to lysosomes and whether such a process might be selective for certain cell proteins are still unresolved. We examined the mechanism of autophagy with isolated autophagic vacuoles. Administration of leupeptin, a inhibitor of lysosomal thiol proteinases, induced the accumulation of numerous autophagic vacuoles in the liver. Highly purified preparation of autophagic vacuoles was isolated by Percoll density gradient equilibrium fractionation of crude lysosomal fractions. When cytosolic enzyme activities in autophagic vacuoles were measured, tyrosine aminotransferase and tryptophan oxygenase with short half-lives, and lactic dehydrogenase and aspartate aminotransferase with long half-lives were detected at similar ratios of enzymes in autophagic vacuoles/cytosol. During the time that cathepsin B plus L activities in autophagic vacuoles are inhibited by the injection of leupeptin, cytosolic enzymes are being accumulated in autophagic vacuoles suggesting that leupeptin blocks intralysosomal proteolysis, and that cytosolic enzymes are sequestered continuously into autophagosomes. Administration of glucocorticoid, which induces the synthesis of tyrosine aminotransferase, tryptophan oxygenase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Dietary manipulation and administration of insulin, which inhibit the formation of autophagic vacuoles, suppressed completely the accumulation of autophagic vacuoles in liver by administration of leupeptin. Results indicate that there is no selective uptake of cytosolic enzymes into autophagosome. When distribution of lysosomal cathepsin B and L in liver, which are inhibited strongly by leupeptin, was examined immunohistochemically, cathepsin L is found only in hepatocytes, but cathepsin B is localized in sinusoidal cells rather than in hepatocytes, suggesting that cathepsin L plays a most important role in intralysosomal proteolysis in hepatocytes.
细胞质蛋白在体内以不同的半衰期被降解。大部分蛋白质被认为主要在自噬泡-溶酶体系统中降解。然而，细胞蛋白质被递送至溶酶体的机制以及这样一个过程是否可能对某些细胞蛋白质具有选择性仍未得到解决。我们用分离的自噬泡研究了自噬机制。亮抑酶肽（一种溶酶体硫醇蛋白酶抑制剂）的给药诱导了肝脏中大量自噬泡的积累。通过对粗溶酶体部分进行Percoll密度梯度平衡分级分离，分离出了高度纯化的自噬泡制剂。当测量自噬泡中的胞质酶活性时，发现自噬泡/胞质溶胶中具有短半衰期的酪氨酸转氨酶和色氨酸加氧酶，以及具有长半衰期的乳酸脱氢酶和天冬氨酸转氨酶的比例相似。在通过注射亮抑酶肽抑制自噬泡中组织蛋白酶B加L的活性期间，胞质酶在自噬泡中积累，这表明亮抑酶肽阻断了溶酶体内的蛋白水解，并且胞质酶持续被隔离到自噬体中。糖皮质激素的给药诱导酪氨酸转氨酶、色氨酸加氧酶和胞质天冬氨酸转氨酶的合成，选择性地以成比例的程度增加了这些酶的隔离。饮食控制和胰岛素的给药抑制自噬泡的形成，通过给予亮抑酶肽完全抑制了肝脏中自噬泡的积累。结果表明，胞质酶没有被选择性摄取到自噬体中。当通过免疫组织化学检查亮抑酶肽强烈抑制的肝脏中溶酶体组织蛋白酶B和L的分布时，发现组织蛋白酶L仅存在于肝细胞中，但组织蛋白酶B定位于窦状细胞而非肝细胞中，这表明组织蛋白酶L在肝细胞的溶酶体内蛋白水解中起最重要的作用。
