title: Purification of an endopeptidase from bovine adrenal medulla granules which cleaves in vitro at paired but not at single basic residues.
abstract: An endopeptidase was isolated from bovine adrenomedullary granules by fast protein liquid chromatography, including two ion exchange, one hydrophobic interaction, and one gel filtration step. The enzyme assay was based on the HPLC detection of the degradation of the dodecapeptide BAM 12P from the sequence of proenkephalin. After a 1200-fold purification, the enzyme fraction was homogeneous on polyacrylamide gel electrophoresis. The apparent molecular weight of the monomeric protein was 68,000 and its pH optimum was 5.6, in agreement with the internal pH of the granules. The specificity of the protease was determined initially by analysis of the degradation fragments of BAM 12P which showed that cleavage occurred at the double but not at the single Arg site of BAM 12P. The cleavage pattern of other substrates showed that the enzyme also recognized other pairs of basic amino acids. The behavior of the enzyme toward specific inhibitors showed that it was an acid thiol protease different from serine proteases and from lysosomal cathepsin B. The endopeptidase may act as a maturation enzyme in vivo.
authors.affiliation: Department of Biotechnology, Swiss Federal Institute of Technology, ETH, Hönggerberg HPT, Zurich, Switzerland.
authors.forename: G E
authors.initials: GE
authors.lastname: Maret
authors.forename: J L
authors.initials: JL
authors.lastname: Fauchère
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D002845:Chromatography / Q000379:methods; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004745:Enkephalins / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology; D008970:Molecular Weight; D011498:Protein Precursors / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D010450:Endopeptidases