Biosynthesis of the multiple forms of rabbit cardiac cathepsin D.
Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.
D000818:Animals; D002402:Cathepsin D; D002403:Cathepsins / Q000096:biosynthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D010477:Perfusion; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology
D007527:Isoenzymes; D002403:Cathepsins; D002402:Cathepsin D