Assessment of the in vivo hepatic lysosomal processing of horseradish peroxidase.
The lysosomal processing of horseradish peroxidase (HRP) was assessed in this study, i.e., its lysosomal proteolysis and the biliary output of its possible lysosomal metabolites by rat liver in vivo. HRP was covalently linked to [14C]sucrose to provide a label that remains trapped within lysosomes after proteolysis. The [14C]sucrose-labelled HRP was injected into the portal vein of rat, and after 30 min about 34% of the injected radiolabel was present in the liver. Subcellular fractionation by differential centrifugation and further purification of lysosomes in a Percoll gradient showed that radiolabel was concentrated in lysosomes and indicated that about 91% of the total proteolysis of HRP in liver could be attributed to these organelles. The in vivo lysosomal degradation rate of HRP at 30 min was about 40%/h, decreasing over time. The lysosomal inhibitors chloroquine and leupeptin suppressed proteolysis of HRP by about 30 and 60%, respectively. Analysis of the 14C excreted in bile by trichloroacetic acid precipitation and by SDS-polyacrylamide gel electrophoresis showed a minor fraction, which was intact HRP (40 kDa), and a major fraction, which was associated with material smaller than 3 kDa. The biliary output of these low molecular mass products, in contrast to that of intact HRP, did not gradually decline with time and represented about 3% of the corresponding amounts in liver. Chloroquine and leupeptin specifically decreased their biliary excretion (about 60%), giving additional support to their lysosomal origin. In addition, the overall hepatic processing of [14C]sucrose-labelled HRP did not differ from that of the native HRP measured by enzyme assay, indicating no significant alteration caused by the labelling procedure.
Marinelli
R A
RA
Instituto de Fisiología Experimental, Universidad Nacional de Rosario, Argentina.
Pellegrino
J M
JM
Larocca
M C
MC
D000818:Animals; D002738:Chloroquine / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D002738:Chloroquine; D006735:Horseradish Peroxidase