Further Characterization of an Enkephalin‐Generating Enzyme from Adrenal Medullary Chromaffin Granules

Abstract

A bovine adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) was purified 1000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent MW of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, pPeptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The 2 heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, peptides E and F were cleaved. The low MW products of peptide F digestion were identified by HPLC [high performance liquid chromatography] as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6Arg7. [3H]-.beta.m-Lipotropin was not hydrolyzed by the adrenal enzyme. This adrenomedullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.