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An Endogenous Aminoenkephalinase Inhibitor: Purification and Characterization of Arg 0 ‐Met 5 ‐Enkephalin from Bovine Striatum
Author(s) -
Hui KoonSea,
Lo EeSing,
Hui Maria PoPing
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.63051748.x
Subject(s) - high performance liquid chromatography , chemistry , peptide , chromatography , fast protein liquid chromatography , enkephalin , biochemistry , receptor , opioid
Arg 0 ‐Met 5 ‐enkephalin (Arg 0 ‐MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroacetic acid, followed by column chromatography successively on Bio‐Sil C 8 , semipreparative HPLC Radial‐Pak C 18 , fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere‐ODS, Supelco C 18 , Lichromsorb C 18 , and μBondapak C 18 . The peptide content was followed by radioimmunoassay with an antibody against synthetic Met‐enkephalin. For each of the six HPLC and FPLC systems, the elution time of the immunoreactive fractions coincided exactly with that of synthetic Arg 0 ‐MEK. The purified peptide showed a highly homogeneous profile in three different analytical HPLC systems. Its retention time and three‐dimensional UV spectrum were identical to those of the synthetic Arg 0 ‐MEK. The structure of the purified material was identified by microsequencing as the hexapeptide Arg‐Tyr‐Gly‐Gly‐Phe‐Met. Ninety percent of the purified peptide was in oxidized form containing equimolar amounts of Met‐( R )‐ and Met‐( S )‐sulfoxide. The reduced Arg 0 ‐MEK inhibited aminoenkephalinase with a K i of 2.2 µ M , and its sulfoxide analogue inhibited it with a K i of 8.9 µ M . The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED 50 of 5 µ M , and the effect could be reversed by equimolar naloxone. Our data indicate that Arg 0 ‐MEK is an immediate Met‐enkephalin precursor and an endogenous inhibitor.