Metorphamide: isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain

Acid acetone extracts of caudate nucleus from bovine brain were found to contain an amidated opioid octapeptide with the following structure: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2. The peptide has been named metorphamide. Bovine metorphamide appears to be derived by proteolytic cleavage from proenkephalin, the common precursor to [Met5]enkephalin and [Leu5]enkephalin. The cleavage within the precursor giving rise to the carboxyl terminus of metorphamide occurs at a single arginine residue and is followed by transformation of a carboxyl-terminal glycine into an amide group. Metorphamide was detected in bovine caudate nucleus extracts by radioimmunoassay, and it was purified to homogeneity by gel filtration and reversed-phase high performance liquid chromatography. Amino acid composition analysis and automated Edman degradation in the gas-phase sequencer confirmed the postulated amino acid sequence. Carboxyl-terminal amidation of bovine metorphamide was shown by stability to carboxypeptidase A digestion and full crossreactivity in a radioimmunoassay that required the carboxyl-terminal amide as part of the recognition site. A synthetic replicate of metorphamide as well as several synthetic analogs were tested for opioid activity in several bioassays and binding assays, and metorphamide was found to have a high mu-binding activity. Metorphamide is the only known naturally occurring opioid peptide that has a high mu-binding activity. The kappa-binding activity is approximately equal to 50% that of the mu-binding activity, but delta-binding activity is negligible.