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) possessin g homology to the yeast Kex2 protease. The presence of PC1/3 and PC2 i n secretory vesicles of bovine adrenal medulla (chromaffin granules) i mplicates their role in the processing the precursors of enkephalin, n europeptide 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, pepsta tin A-agarose, and anti-PC1/3 or anti-PC2 immunoaffinity resins. PC1/3 and PC2 were monitored during purification by measuring proteolytic a ctivities with S-35-enkephalin precursor and Boc-Arg-Val-Arg-Arg-methy lcoumarin amide (MCA) substrates and by following PC1/3 and PC2 immuno reactivity with specific anti-PC1/3 and anti-PC2 sera generated in thi s 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 g ranules was present at 5- and 10-fold higher enzyme protein and activi ty, respectively, compared with that of PC1/3. PC1/3 and PC2 cleaved p aired basic and monobasic sites within peptide-MCA substrates, with Bo c-Arg-Val-Arg-Arg-MCA and pGlu-Arg-Thr-Lys-Arg-MCA as the most effecti vely cleaved peptides tested. PC1/3 and PC2 showed pH optima of 6.5 an d 7.0, respectively. Kinetic studies indicated apparent K-m values for hydrolysis of Boc-Arg-Val-Arg-Arg-MCA as 66 and 40 mu M, with V-max v alues of 255 and 353 nmol/h/mg for PC1/3 and PC2, respectively. Specif icity of the PC enzymes for dibasic sites was confirmed by potent inhi bition by the active site-directed peptide inhibitors (D-Tyr)-Glu-Phe- Lys-Arg-CH2Cl and Ac-Arg-Arg-Ch(2)Cl. Inhibition by EGTA and activatio n by Ca2+ indicated PC1/3 and PC2 as Ca2+-dependent proteases. In addi tion, PC enzymes were activated by dithiothreitol and inhibited by thi ol-blocking reagents, p-hydroxymercuribenzoate and mercuric chloride. These results illustrate the properties of endogenous PC1/3 and PC2 as prohormone-processing enzymes.