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 macroautophagy and bulk proteolysis set out and progress. Separation of these particles in freshly isolated rat hepatocytes, monitored by beta-hexosaminidase, a lysosomal marker enzyme, was established by density gradient centrifugation. Percoll density gradients were modified and improved by adding free polyvinylpyrrolidone (PVP, 0.75%) to 60% Percoll, which made it possible to separate AVd (buoyant peak, d = 1.090) and DB (dense peak, d = 1.131) effectively. Addition of graded levels of a regulatory amino acid mixture (Reg AA) to hepatocyte incubation not only suppressed proteolysis, but also lead to a shift of vacuolar profiles on the density gradients from the buoyant to the dense region. Alterations in the vacuolar shift and proteolysis were highly proportional over a full range of regulation by Reg AA. Morphometric analysis of autophagic vacuoles by electron microscopy revealed changes in the aggregate volumes of both AVi and AVd by Reg AA, which enabled us to estimate autophagic 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 gradient profile can be used as a measure of macroautophagy; and in addition that AVd actively involved in proteolysis occupies only a part of the buoyant peak on the gradients.