Processing of Proenkephalin in Adrenal Chromaffin Cells

The processing of proenkephalin was studied using 35S]methionine pulse-chase techniques in primary cultures of bovine adrenal medullary chromaffin cells. Following radiolabeling, proenkephalin-derived peptides were extracted from the cells and separated by reverse-phase HPLC. Fractions containing proenkephalin fragments were digested with trypsin and carboxypeptidase B to liberate Met-enkephalin sequences and subjected to a second HPLC step to demonstrate association of radiolabel with Met-enkephalin. Processing of proenkephalin is complete within 2 h of synthesis, suggesting completion at or soon after incorporation into storage vesicles. Pretreatment of the cells with nicotine, histamine, or vasoactive intestinal peptide to enhance the rate of proenkephalin synthesis failed to alter the time course of processing and had minimal effects on the distribution of products formed. Addition of tetrabenazine, an inhibitor of catecholamine uptake into chromaffin vesicles, during radiolabeling and a 6-h chase period caused enhanced proenkephalin processing. These results suggest that the full range of proenkephalin fragments normally found in the adrenal medulla (up to 23.3 kDa) represents final processing products of the tissue and that termination of processing may depend on the co-storage of catecholamines.