title: Transport and proteolytic processing of rabbit cardiac cathepsin D.
abstract: Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex; D001692:Biological Transport; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology; D007106:Immune Sera; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008854:Microscopy, Electron; D009206:Myocardium / Q000201:enzymology* / Q000648:ultrastructure; D010447:Peptide Hydrolases / Q000378:metabolism*; D010477:Perfusion; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology; D013462:Sulfur Radioisotopes
chemical_list: D000936:Antigen-Antibody Complex; D007106:Immune Sera; D013462:Sulfur Radioisotopes; D002738:Chloroquine; D010447:Peptide Hydrolases; D002402:Cathepsin D