An endogenous aminoenkephalinase inhibitor: purification and characterization of Arg0-Met5-enkephalin from bovine striatum.
Arg0-Met5-enkephalin (Arg0-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 C8, semipreparative HPLC Radial-Pak C18, fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C18, Lichromsorb C18, and mu Bondapak C18. 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 Arg0-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 Arg0-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 Arg0-MEK inhibited aminoenkephalinase with a Ki of 2.2 microM, and its sulfoxide analogue inhibited it with a Ki of 8.9 microM. The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED50 of 5 microM, and the effect could be reversed by equimolar naloxone. Our data indicate that Arg0-MEK is an immediate Met-enkephalin precursor and an endogenous inhibitor.
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Neurochemistry Division, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York 10962.
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003342:Corpus Striatum / Q000737:chemistry*; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011863:Radioimmunoassay
D004744:Enkephalin, Methionine; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme