Identification of autolysosomes directly associated with proteolysis on the density gradients in isolated rat hepatocytes

Autophagy-related vacuoles, i.e., autophagosomes (AVi), autolysosomes (AVd) and dense bodies (DB), are intracellular organelles within which macroauto phagy and bulk proteolysis set out and progress. Separation of these partic les in freshly isolated rat hepatocytes, monitored by P-hexosaminidase, a l ysosomal marker enzyme, was established by density gradient centrifugation, Percoll density gradients were modified and improved by adding free polyvi nylpyrrolidone (PVP, 0.75%) to 60% Percoll, which made it possible to separ ate AVd (buoyant peak, d=1.090) and DB (dense peak, d=1.131) effectively. A ddition of graded levels of a regulatory amino acid mixture (Reg AA) to hep atocyte incubation not only suppressed proteolysis, but also lead to a shif t of vacuolar profiles on the density gradients from the buoyant to the den se region. Alterations in the vacuolar shift and proteolysis were highly pr oportional over a full range of regulation by Beg AA, Morphometric analysis of autophagic vacuoles by electron microscopy revealed changes in the aggr egate volumes of both AVi and AVd by Beg AA, which enabled us to estimate a utophase subpopulation of the buoyant peak on the gradient profile, All the results demonstrate that AVd shifts on the density gradients in proportion to alterations in proteolysis regulated by amino acids, and thus the gradi ent profile can be used as a measure of macroautophagy; and in addition tha t AVd actively involved in proteolysis occupies only a part of the buoyant peak on the gradients.