Characterization of a metalloprotease from ovine chromaffin granules which cleaves a proenkephalin fragment (BAM12P) at a single arginine residue.
A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions.
Tezapsidis
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Unit of Metabolic Medicine, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, Paddington, London, U.K.
Parish
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D000313:Adrenal Medulla / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002413:Cations, Divalent; D002837:Chromaffin Granules / Q000201:enzymology*; D002845:Chromatography; D003300:Copper / Q000494:pharmacology; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism* / Q000494:pharmacology; D012756:Sheep; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D004745:Enkephalins; D010446:Peptide Fragments; D011498:Protein Precursors; C029992:proenkephalin; D004744:Enkephalin, Methionine; D003300:Copper; C039700:adrenorphin; D001120:Arginine; D008666:Metalloendopeptidases; D015032:Zinc