Rapid isolation of rat liver secondary lysosomes—Autophagic vacuoles—Following chloroquine administration

Abstract

A technique is presented for the rapid isolation of secondary lysosomes—autophagic vacuoles (AVs)—from rat liver by a one-step centrifugation in a discontinuous Metrizamide gradient. To this end chloroquine was injected into rats in order to increase the number of AVs, a prerequisite for their isolation, since they are rare in control liver tissue. Fraction purity was some 85–90% as judged from morphological analyses. To assess the proteolytic ability of the AVs they were isolated from livers of rats injected with [ 14C]leucine. Proteolysis increased significantly peaking after 60 min of chloroquine exposure, by far exceeding values for lysosomes isolated from control livers. This is in contrast to AVs (secondary lysosomes) obtained after leupeptin treatment which display lowered proteolysis as compared with control. After longer exposure times to chloroquine, proteolysis gradually returned to basal level. It is surmised that the augmented proteolysis in the isolated AVs is due to increased contents of substrate. So far, the chloroquine-induced AVs (secondary lysosomes) seem to be the best candidates for further analyses of proteolytic events in these organelles.