Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9.
1. The kinetics of the degradation of the kinins bradykinin and Met-Lys-bradykinin, angiotensins I and II and the tachykinin substance P by PMNL-collagenase (MMP 8), PMNL-gelatinase (MMP 9) and by the recombinant catalytic domain of MMP 8 (rcd-PMNL-c) was examined by RP-HPLC. The resulting fragments were identified by automated Edman degradation or by amino acid analysis. 2. The initial degradation rates of substance P at a substrate concentration of 25 microM were 5 min-1 for MMP 9 and 150 min-1 for MMP 8. The kinetic constants KM and kcat were determined by concentration-dependent measurements. For MMP 8/substance P the constants were KM = 78 +/- 14 microM and kcat = 412 +/- 67 min-1. For MMP 9/substance P the constants were KM = 91 +/- 15 microM and kcat = 25 +/- 4 min-1. Both enzymes cleaved substance P between Gln6 and Phe7 and between Gly9 and Leu10. 3. Under the same conditions, MMP 8 degraded angiotensin I at an initial rate of 20 h-1, resulting mainly in the vasoactive fragments angiotensin II and angiotensin(1-7). At a substrate concentration of 25 microM and an enzyme/substrate ratio of 1:100, angiotensin II was degraded very slowly (19% in 24 h) by MMP 8. Under these conditions, MMP 9 degraded angiotensin I to a lesser extent than MMP 8 (25% in 24 h) and was unable to cleave angiotensin II. 4. Under the same conditions, bradykinin and Met-Lys-bradykinin were cleaved by PMNL-collagenase at a rate of 20% in 24 h, producing BK(1-7) and BK(1-8). PMNL-gelatinase was unable to cleave the kinins under these conditions. 5. In all cases, rcd-PMNL-c produced the same fragments as wild type PMNL-collagenase, but at a significantly lower rate.
Diekmann
O
O
Department of Biochemistry, Faculty of Chemistry, University of Bielefeld, Germany.
Tschesche
H
H
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000809:Angiotensins / Q000378:metabolism*; D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D017364:Collagenases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D007705:Kinins / Q000378:metabolism*; D020784:Matrix Metalloproteinase 8; D020780:Matrix Metalloproteinase 9; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D013373:Substance P / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
D000809:Angiotensins; D007705:Kinins; D010446:Peptide Fragments; D000804:Angiotensin II; D013373:Substance P; C018103:bradykinin, Met-Lys-; D000803:Angiotensin I; D017364:Collagenases; D020784:Matrix Metalloproteinase 8; D020780:Matrix Metalloproteinase 9; D001920:Bradykinin