Limited proteolysis of rabbit cardiac procathepsin D in a cell-free system.
Rabbit cardiac cathepsin D is initially synthesized as an inactive, apparent molecular weight (Mr) 53,000, pI 6.6 precursor (procathepsin D) that is proteolytically processed during intracellular transport to produce the Mr 48,000 isoforms of active cathepsin D found in cardiac lysosomes. To examine potential proteases responsible for intracellular proteolytic processing, biosynthetically labeled procathepsin D was isolated from rabbit ventricular tissue perfused for 30 min with [35S]methionine. Procathepsin D was then incubated in vitro (40 degrees C, 1-240 min) with active cathepsin D, papain, and cathepsin B, either singly or sequentially, and the reaction products analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Incubation of 35S-labeled procathepsin D with active cathepsin D produced a single reaction product (Mr 51,000; pI 6.2). This limited proteolysis occurred at pH 3-5 and was inhibited by pepstatin. Incubation of 35S-labeled procathepsin D with papain or cathepsin B produced a major reaction product (Mr 48,000; pI 6.4) and a minor form (Mr 50,000; pI 6.0). These reactions occurred at pH 4-7 and were inhibited by leupeptin but not pepstatin. Only the Mr 48,000, pI 6.4 products of papain and cathepsin B-mediated proteolysis comigrated with the most basic isoform of active cathepsin D found in cardiac tissue. In addition, the Mr 51,000 intermediate produced by cathepsin D was susceptible to further limited proteolysis by cysteine proteases with resultant production of a Mr 48,000 product. Thus the intracellular proteolytic processing of rabbit cardiac procathepsin D does not result solely from autocatalysis but requires at least one other protease, possibly cathepsin B.
D000818:Animals; D002401:Cathepsin B; D002402:Cathepsin D / Q000378:metabolism*; D002403:Cathepsins / Q000378:metabolism; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism*; D005470:Fluorometry; D007526:Isoelectric Point; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010206:Papain / Q000378:metabolism; D011817:Rabbits
D004792:Enzyme Precursors; D008715:Methionine; D002403:Cathepsins; D002401:Cathepsin B; D010206:Papain; D002402:Cathepsin D