Cleavage of recombinant enkephalin precursor by endoproteolytic activity in bovine chromaffin granules.

To identify endoproteolytic activity that processes the enkephalin precursor, a novel approach was undertaken for the production of model substrate in the form of recombinant 35S-(Met)-preproenkephalin (35S-(Met)-PPE), generated by in vitro transcription and translation of the rat PPE cDNA. Endoproteolytic activity in bovine chromaffin granules cleaved 35S-(Met)-PPE with a pH optimum of 4.5 and generated multiple products containing the NH2-terminal segment of the precursor. Processing of 35S-(Met)-PPE, as well as endogenous enkephalin intermediates, was inhibited by pepstatin A and stimulated by DTT. These results suggest that aspartyl and thiol proteolytic activity(ies) are involved in cleaving the enkephalin precursor.