Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin.

Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later.