Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates

Naoko Kojima, Shun-Ichiro Kawabata, Yuichi Makinose, Norikazu Nishino, Sadaaki Iwanaga

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Abstract

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.

Original languageEnglish
Pages (from-to)855-861
Number of pages7
JournalJournal of biochemistry
Volume118
Issue number4
DOIs
Publication statusPublished - Jan 1 1995

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Metalloendopeptidases
Substrate Specificity
Fluorescent Dyes
Liver
thimet oligopeptidase
Rabbits
Peptides
Substrates
Oligopeptides
Bradykinin
Cytosol
Angiotensins
phenylalanylserine
Hydrolysis
Low Molecular Weight Kininogens
para-Aminobenzoates
Endorphins
Angiotensin I
Neurotensin
Adrenal Medulla

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates. / Kojima, Naoko; Kawabata, Shun-Ichiro; Makinose, Yuichi; Nishino, Norikazu; Iwanaga, Sadaaki.

In: Journal of biochemistry, Vol. 118, No. 4, 01.01.1995, p. 855-861.

Research output: Contribution to journalArticle

Kojima, Naoko ; Kawabata, Shun-Ichiro ; Makinose, Yuichi ; Nishino, Norikazu ; Iwanaga, Sadaaki. / Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates. In: Journal of biochemistry. 1995 ; Vol. 118, No. 4. pp. 855-861.
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N2 - 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.

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