AN ENDOGENOUS AMINOENKEPHALINASE INHIBITOR - PURIFICATION AND CHARACTERIZATION OF ARG(0)-MET(5)-ENKEPHALIN FROM BOVINE STRIATUM

Arg(0)-Met(5)-enkephalin (ArS0-MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroa cetic acid, followed by column chromatography successively on Bio-Sil C-8, semipreparative HPLC Radial-Pak C-18, fast protein liquid chromat ography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C-18, Lichromsorb C-18, and mu Bondapak C-18. The peptide content was followed by radio immunoassay with an antibody against synthetic Met-enkephalin. For eac h of the six HPLC and FPLC systems, the elution time of the immunoreac tive fractions coincided exactly with that of synthetic Arg(0)-MEK. Th e purified peptide showed a highly homogeneous profile in three differ ent analytical HPLC systems. Its retention time and three-dimensional UV spectrum were identical to those of the synthetic Arg(0)-MEK. The s tructure of the purified material was identified by microsequencing as the hexapeptide Arg-Tyr-Gly-Gly-Phe-Met. Ninety percent of the purifi ed peptide was in oxidized form containing equimolar amounts of Met-(R )- and Met-(S)-sulfoxide. The reduced Arg(0)-MEK inhibited aminoenkeph alinase with a K-i of 2.2 mu M, and its sulfoxide analogue inhibited i t with a K-i of 8.9 mu M. The reduced or oxidized peptide suppressed t he electrically induced contraction of rat vas deferens with an ED(50) of 5 mu M, and the effect could be reversed by equimolar naloxone. Ou r data indicate that Arg(0)-MEK is an immediate Met-enkephalin precurs or and an endogenous inhibitor.