THE POSTTRANSLATIONAL PROCESSING AND INTRACELLULAR SORTING OF CARBOXYPEPTIDASE-H IN THE ISLETS OF LANGERHANS

The post-translational processing and intracellular sorting of the pro insulin-converting enzyme carboxypeptidase H (CPH) was studied in isol ated rat islets of Langerhans. Pulse-chase-radiolabelling experiments using sequence-specific antisera showed that CPH was synthesized initi ally as a 57-kDa glycoprotein which was processed to a 54-kDa mature f orm by proteolytic processing at the N-terminus. Processing of the CPH precursor occurred rapidly (t(1/2) = 30 min) after an initial delay o f 15-30 min and the enzyme was secreted in parallel with the insulin-r elated peptides in response to glucose-stimulation within 1 h after ra diolabelling. This indicated that the proteins were packaged into nasc ent secretory granules at approximately the same rate following synthe sis. Conversion of proinsulin and the 57-kDa form of CPH was inhibited markedly by chase incubation of islets at 20 degrees C, indicating th at maturation of both proteins occurs in a post-Golgi compartment. Aff inity purification of the enzyme from insulinoma subcellular fractions showed that the 57-kDa form was associated with endoplasmic reticulum or Golgi elements, and the 54-kDa form was present in secretory granu les. Structural analysis showed that the granule form of the enzyme ha d an N-terminal amino acid sequence beginning at residue 42 of rat CPH , thereby implicating cleavage of the precursor after the fourth Arg i n a site containing five consecutive Arg residues. These findings indi cate that post-translational processing of CPH is mediated by an endop rotease which cleaves at sites containing multiple basic amino acid re sidues upon segregation of the enzyme to the secretory granules.