Synaptosomal Membrane‐Bound Form of Endopeptidase‐24.15 Generates Leu‐Enkephalin from Dynorphin 1‐8, α‐ and β‐Neoendorphin, and Met‐Enkephalin from Met‐Enkephalin‐Arg6‐Gly7‐Leu

Abstract

Brain contains a membrane-bound form of endopeptidase-24. 15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20–25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endo-peptidase-24.11 (“enkephalinase”), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphini_1-8, α-and β-neoen-dorphin into leucine enkephalin and methionine-enkepha-Iin-Arg⁶-Gly⁷-Leu⁸ into methionine enkephalin in the presence of captopril, bestatin, and N-[l- (R, S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[l- (R, S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine-and methionine-enkephalin.