Processing of proenkephalin in bovine chromaffin cells occurs in two phases

The processing of proenkephalin was studied in primary cultures of bovine adrenal medullary chromaffin cells by pulse-chase radiolabeling, immunopurification of proenkephalin and derivative peptides and quantitation following gel electrophoresis and Western blotting. Proenkephalin was processed with a t1/2 of approximately 1.1 h. Processing of proenkephalin-derived peptides of 15-25 kDa was essentially complete by 1 h. Treatment of chromaffin cells with brefeldin A to block the intracellular transport of proteins or with ammonium chloride to neutralize acidic intracellular compartments had only minor effects on the initial processing of proenkephalin. In contrast, both of these agents prevented a second, slower phase of proenkephalin processing. These studies suggest that proteolytic processing of proenkephalin in bovine adrenal medullary cells starts before transport to the trans-Golgi network and packaging into the chromaffin granules. A second phase of processing that requires an acidic environment occurs in or distal to the trans-Golgi network.