A metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol. Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme. MEP degraded oligopeptides, including bradykinin, α-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bom-besin, neurotensin, and α-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases. It cleaved most preferentially at the -Phe-Ser- bond of bradykinin(kcat/Km=2.8×104M-1 s-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin. MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibbasic convertase, and yeast endopeptidase-24.15,related peptidase. An active site-directed inhibitor of metalloendo-peptidase- 24.15, N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP. Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized. Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-2, 4-dinitroanilinoethylamide(kcat/=9.3×105M-1S-1). An angiotensin antagonist, [Sar1, Ala8] -angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (K1 = 7.6 μM).MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.
|Number of pages||7|
|Journal||Journal of biochemistry|
|Publication status||Published - Jan 1 1995|
All Science Journal Classification (ASJC) codes
- Molecular Biology