Comparative incorporation of proenkephalin-derived peptides, chromogranin A, and dopamine β-hydroxylase into chromaffin vesicles

The incorporation of enkephalin-containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse-labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met-enkephalin sequences in gradient fractions determined. 35S-ECPs were incorporated into particles suggestive of immature vesicles within 1–2 h after radiolabeling. Vesicle maturation, measured by co-equilibration of 35S-ECPs and total ECPs in the gradients, was complete within 9–12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S-labeled dopamine β-hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.