Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.
Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.
Kopitz
J
J
Physiological-Chemical Institute, University of Tübingen, Federal Republic of Germany.
Kisen
G O
GO
Gordon
P B
PB
Bohley
P
P
Seglen
P O
PO
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000201:enzymology*; D004560:Electricity; D005260:Female; D006207:Half-Life; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure
D007976:Leupeptins; D007770:L-Lactate Dehydrogenase; C032854:leupeptin