Purification and characterization of a secreted arginine-specific dibasic cleaving enzyme from EL-4 cells.
A secreted dibasic cleaving peptidase capable of converting dynorphins into Leu-enkephalin-Arg6 was purified from the medium of EL-4 mouse thymoma cells. The enzyme is a novel metalloendopeptidase with a neutral pH optimum (6.9) and a molecular weight of approximately 130 000. The dibasic cleaving enzyme was completely inhibited in the presence of 20-50 mM amine buffers, 0.1 mM EDTA, 0.5 mM 1,10-phenanthroline, 0.5 mM N-ethylmaleimide, and 1mM DTNB. Unlike the Kex2 family of proteases, Ca2+ did not activate the endopeptidase, but high concentrations (1 mM) of metal ions such as Cu2+, Ni2+, Zn2+, and Co2+ completely inhibited the enzyme. Inhibition was not seen with 0.2 mM TLCK, 1 mM DTT, and 1 mM PMSF. The enzyme will cleave Arg-Arg and Arg-Lys bonds, but not Lys-Arg or Lys-Lys bonds in identical environments, and no aminopeptidase or carboxypeptidase activity was seen. The size of the substrate does not seem to be a determining factor, since dynorphin A(1-12) is cleaved at a rate similar to prodynorphin B(228-256) containing 29 amino acids. The identity of the residues on either side of the cleavage site influences the rate of processing, as noted by different rates of cleavage for the same size peptides dynorphin A(1-13) vs dynorphin A(1-9) vs beta-neoendorphin. The presence of proline in the P3' (alpha-neoendorphin), P4' (dynorphin A(1-11)), or P5' (bovine adrenal medulla dodecapeptide) position does not prevent cleavage, but neurotensin and its (1-11) fragment containing both P2 and P2' proline residues are not cleaved.
Csuhai
E
E
Department of Biochemistry, University of Kentucky, Lexington 40536, USA.
Safavi
A
A
Hersh
L B
LB
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D002413:Cations, Divalent / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D004399:Dynorphins / Q000378:metabolism*; D004723:Endorphins / Q000378:metabolism*; D004791:Enzyme Inhibitors; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D011480:Protease Inhibitors; D011498:Protein Precursors / Q000378:metabolism; D013379:Substrate Specificity; D013945:Thymoma / Q000201:enzymology; D014325:Tromethamine / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D004723:Endorphins; D004791:Enzyme Inhibitors; D008670:Metals; D011480:Protease Inhibitors; D011498:Protein Precursors; D014325:Tromethamine; C034249:1,3-bis(tris(hydroxymethyl)methylamino)propane; C025681:alpha-neoendorphin; D004399:Dynorphins; C036200:rimorphin; D001120:Arginine; D003546:Cysteine Endopeptidases; C063078:dynorphin converting enzyme; D008666:Metalloendopeptidases; C087976:nardilysin