Peptide Intermediates in the Degradation

W e have previously shown that bestatin (an aminopeptidase inhibitor) permits the accumulation of diand tripeptide intermediates in the degradation of abnormal globin (Botbol, V., and Scornik, 0. A. (1979) Proc. Natl. Acad. Sei. U. S. A. 76, 710-713; Botbol, V., and Scornik, 0. A. (1979) J. Biol. Chem. 254, 11254-11257). We now report that this drug (1 mg, intravenously, 5-30 min) causes similar intermediates to appear during breakdown of normal cellular proteins in the livers of live mice labeled 1 or 20 h before with ~ [ l ’~C]Leu or ~-[l-”C]Arg. These intermediates represent an estimated 20% of all degraded cellular protein. Lysosomal degradation of labeled asialoglycoproteins taken up by endocytosis is less affected by bestatin. After homogenization and centrifugation of the livers, we find a major fraction of the intermediates in the cytosol. Another fraction sediments-at 27,000 X g in 15 min. The fraction that sediments is larger for Arg-labeled (30%) than for Leu-labeled (10%) peptides. The particulate fraction does not represent soluble peptides trapped in the pellet during fractionation because it does not appear when soluble intermediates are added to nonradioactive homogenates. The presence of intermediates in particulate and soluble fractions could result from protein breakdown either in both compartments or in organelles from which the peptides escape. We favor the latter possibility because (a) the particulate fraction does not increase after stimulation of autophagy by injection of glucagon (40 pg, 75 min before killing), (b) the subcellular distribution is the same whether intermediates are produced during the degradation of short or long lived proteins, and (e) chromatographic fingerprints of the particulate and soluble components reveal the same seven bands. The presence of well defined intermediates of cellular protein degradation in the particulate fraction of liver homogena tes provides a valuable marke r in the isolation a n d characterization of autophagic organelles.