Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.