SEPARATION OF LYSOSOMES AND AUTOPHAGOSOMES BY MEANS OF GLYCYL-PHENYLALANINE-NAPHTHYLAMIDE, A LYSOSOME-DISRUPTING CATHEPSIN-C SUBSTRATE

In density-gradient analyses of autophagic vacuoles from isolated rat hepatocytes, autophagosomes could be recognized by the presence of an autophagically sequestered cytosolic enzyme, lactate dehydrogenase (LD H). Lysosomes were identified by marker enzymes such as acid phosphata se, or by degradation products from I-125-tyramine-cellobiose-asialoor osomucoid (I-125-TC-AOM) loaded into the lysosomes by an intravenous i njection in vivo 18 h prior to cell isolation. Autophagosomes and lyso somes showed similar, largely overlapping, density distributions both in hypertonic sucrose gradients and in isotonic Nycodenz gradients. As a step towards the purification of autophagosomes, we investigated th e possibility of using lysosomal enzyme substrates to achieve selectiv e destruction of lysosomes by swelling. Hepatocytes were first incubat ed for 2h at 37 degrees C with vinblastine (50 mu M) to obtain an accu mulation of autophagosomes (to 3-5-times above the control level). The cells were then electrodisrupted and the disruptates incubated with a variety of substrates for lysosomal enzymes. Among these, glycyl-phen ylalanine-2-naphthylamide (GPN), a cathepsin-C substrate, and methioni ne-O-methylester (MetOMe), an esterase substrate, turned out to induce extensive rupture of lysosomes, as measured by a strongly reduced sed imentability of acid phosphatase and a nearly complete loss of I-125-T C-AOM sedimentability in substrate-treated preparations from control o r vinblastine-treated cells. The lysosomes of cells treated with leupe ptin or asparagine were largely resistant to the action of GPN, probab ly as a result of interference with cathepsin-C activity or lysosomal function in general. Autophagosomes were partially destroyed by MetOMe , as indicated by a reduction in sedimentable LDH, but GPN had no effe ct on either autophagosomes or mitochondria. The ability of GPN to sel ectively destroy lysosomes without affecting the autophagosomes of vin blastine-treated cells should make GPN treatment a useful aid in the p urification of rat liver autophagosomes.