title: Cleavage of recombinant enkephalin precursor by endoproteolytic activity in bovine chromaffin granules.
abstract: 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.
authors.affiliation: Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.
authors.forename: V Y
authors.initials: VY
authors.lastname: Hook
authors.forename: D
authors.initials: D
authors.lastname: Hegerle
authors.forename: H U
authors.initials: HU
authors.lastname: Affolter
mesh_terms: D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D004745:Enkephalins / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004745:Enkephalins; D011480:Protease Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; C033660:preproenkephalin; D010447:Peptide Hydrolases