Purification and characteristics of the candidate prohormone processing proteases PC2 and PC1/3 from bovine adrenal medulla chromaffin granules.

The prohormone-processing proteases PC1/3 and PC2 belong to the family of mammalian subtilisin-related proprotein convertases (PC) possessing homology to the yeast Kex2 protease. The presence of PC1/3 and PC2 in secretory vesicles of bovine adrenal medulla (chromaffin granules) implicates their role in the processing the precursors of enkephalin, neuropeptide Y, somatostatin, and other neuropeptides that are present in chromaffin granules. In this study, PC1/3 and PC2 were purified to apparent homogeneity from the soluble fraction of chromaffin granules by chromatography on concanavalin A-Sepharose, Sephacryl S-200, pepstatin A-agarose, and anti-PC1/3 or anti-PC2 immunoaffinity resins. PC1/3 and PC2 were monitored during purification by measuring proteolytic activities with 35S-enkephalin precursor and Boc-Arg-Val-Arg-Arg-methylcoumarin amide (MCA) substrates and by following PC1/3 and PC2 immunoreactivity with specific anti-PC1/3 and anti-PC2 sera generated in this study. Purified PC1/3 and PC2 on SDS-polyacrylamide gels each show a molecular mass of 66 kDa. PC2 in the soluble fraction of chromaffin granules was present at 5- and 10-fold higher enzyme protein and activity, respectively, compared with that of PC1/3. PC1/3 and PC2 cleaved paired basic and monobasic sites within peptide-MCA substrates, with Boc-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most effectively cleaved peptides tested. PC1/3 and PC2 showed pH optima of 6.5 and 7.0, respectively. Kinetic studies indicated apparent Km values for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 microM, with Vmax values of 255 and 353 nmol/h/mg for PC1/3 and PC2, respectively. Specificity of the PC enzymes for dibasic sites was confirmed by potent inhibition by the active site-directed peptide inhibitors (D-Tyr)-Glu-Phe-Lys-Arg-CH2Cl and Ac-Arg-Arg-CH2Cl. Inhibition by EGTA and activation by Ca2+ indicated PC1/3 and PC2 as Ca(2+)-dependent proteases. In addition, PC enzymes were activated by dithiothreitol and inhibited by thiol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride. These results illustrate the properties of endogenous PC1/3 and PC2 as prohormone-processing enzymes.