Degradation of short- and long-lived proteins in perfused liver and in isolated autophagic vacuoles--lysosomes.

The lysosomal contribution to breakdown of prelabeled short- and long-lived cell proteins in the perfused rat liver was monitored in two ways. In the first, either leupeptin or chloroquine was injected into rats as well as added to the perfusate. The extent of suppression of proteolysis compared to that of controls was equated with the lysosomal share of protein breakdown. Both compounds inhibited degradation of short-lived proteins by some 30% and long-lived proteins by some 60%. In the second approach, lysosomal-autophagic vacuolar (LAV) fractions were isolated from livers pretreated and perfused as above. The LAV fractions generated different amounts of degradation products during subsequent incubations. They were, in decreasing order of magnitude, that of the LAV fraction isolated from livers perfused with chloroquine, without inhibitor, and with leupeptin. The LAV fraction from control livers was considered to yield the most reliable estimate of the lysosomal share of proteolysis as recorded during liver perfusions. It was 54% for short-lived proteins and 75% for long-lived proteins. These figures are higher than those obtained in the perfusion experiments in the first approach. It is therefore suggested that quantitative assessment concerning the lysosomal share of overall proteolysis may be underestimated when based on inhibition experiments performed on intact cells. The ultrastructure of hepatocytes and LAV fractions was also investigated. Leupeptin-treated hepatocytes displayed more frequent and larger AVs than those of control livers. Cell vacuolation was an additional characteristic of chloroquine-exposed hepatocytes. The LAV fractions isolated from control and leupeptin-treated livers mirrored the observations made in liver tissue. The constituents of the LAV fraction isolated from chloroquine-treated tissue were, however, less swollen than in situ, probably due to the extraction of water during isolation.