Degradation of short and long lived proteins in isolated rat liver lysosomes. Effects of pH, temperature, and proteolytic inhibitors.
Most previous studies on inhibitors of lysosomal protein breakdown have been performed on isolated or cultured cells or on perfused organs. We have tested various inhibitors of proteolysis on lysosomes isolated from livers of rats injected with [14C]leucine 15 min (short labeling time) and 16 h (long labeling time) before killing. Intact lysosomes were incubated with different inhibitors (leupeptin, propylamine, E-64, pepstatin, and chloroquine) in increasing concentrations. None of these caused more than a 40-75% inhibition of proteolysis irrespective of labeling protocol. Chloroquine was the most effective inhibitor, followed by leupeptin, propylamine, E-64, and pepstatin. When lysosomes were incubated with various combinations of inhibitors, including a weak base and an enzyme inhibitor, a somewhat higher inhibition (86%) was obtained. To assess if lysosomes are active in the degradation of both short and long lived proteins, lysosomes were isolated from livers of rats labeled with [14C]leucine for various time intervals. The highest fractional proteolytic rates were seen for short lived proteins. If the recovery of the isolated lysosomes is taken into consideration, about 80% (short labeling time) and 90% (long labeling time) of the total proteolysis in the homogenate could be accounted for by lysosomes. Isolated Golgi, mitochondrial, and microsomal fractions displayed negligible proteolytic activities. The cytosol contributed one-fifth of the total protein breakdown of short lived proteins, whereas insignificant proteolysis was recovered in the cytosolic fraction following long time labeling. Accordingly, we propose that 1) lysosomal inhibitors do not completely suppress proteolysis in isolated lysosomes and that 2) both short and long lived proteins are degraded in lysosomes.
D000818:Animals; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000648:ultrastructure*; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D011480:Protease Inhibitors / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D013997:Time Factors
D011480:Protease Inhibitors; D011506:Proteins