PROCESSING OF PROCARBOXYPEPTIDASE-E INTO CARBOXYPEPTIDASE-E OCCURS INSECRETORY VESICLES

Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor (''proCPE'') that undergoes posttran slational processing at a site containing five adjacent Arg residues n ear the N-terminus and at other sites near the C-terminus of proCPE. T he time course of the N-terminal processing step suggests that this co nversion occurs in either the Golgi apparatus or the secretory vesicle s. To delineate further the site of proCPE processing, pulse/chase ana lysis was performed under conditions that block transit out of the Gol gi apparatus (brefeldin A, carbonyl cyanide m-chlorophenylhydrazone, o r 20 degrees C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compar tment. To test whether known processing enzymes can perform this cleav age, purified proCPE was incubated with furin, prohormone convertase 1 , or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-term inal region, although the reaction is not very efficient, requiring re latively large amounts of furin or long incubation times. The other tw o peptide processing enzymes did not cleave proCPE, whereas a relative ly small amount of secretory granule extract was able to convert proCP E into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.