Characterization of Subfractions of Autolysosomes Isolated from Rat Liver after Leupeptin Treatment

Abstract

Autolysosomes were isolated from leupeptin-treated rat liver (Furuno, K., Ishikawa, T., & Kato, K. (1982) J. Biochem. 91, 1943-1950). They were disrupted by hypotonic treatment and subfractionated by centrifugation in a discontinuous sucrose gradient into three distinct parts: the membranes, the soluble contents, and the insoluble remnants. The content fraction contained the bulk of the activities of lysosomal enzymes with a protein yield of about 36%. The membrane fraction, representing about 5% of protein of the autolysosomes, had a high specific activity of acid phosphatase. In SDS-polyacrylamide gel electrophoretic analysis, the autolysosomal membranes showed protein profiles similar to those of normal lysosomal membranes. The aggregates of partially digested cellular components, including organelles, in the autolysosomes were recovered as granular materials with a very high density (designated as the remnant fraction). This fraction accounted for more than half of protein of the autolysosomes but contained little of the activities of lysosomal enzymes. Lipid analyses revealed that the autolysosomes were poor in lipids because the lipid content of the insoluble remnants was very low. Measurements of the rate of protein degradation in vitro in the crude lysosomal fraction and the isolated autolysosomes from leupeptin-treated rat liver showed that proteolysis was suppressed within the autolysosomes. It was suggested that lipids of sequestered cellular components were preferentially digested within the autolysosomes due to the inhibition of proteolytic activity by leupeptin, and the resulting massive accumulation of proteins was responsible for the enhanced autolysosomal density.