PUB - Publikationen an der Universität Bielefeld

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 mu M were 5 min(-1) for MMP 9 and 150 min(-1) for MMP 8. The kinetic constants K-M and k(cat) were determined by concentration-dependent measurements. For MMP 8/substance P the constants were K-M = 78 +/- 14 mu M and k(cat) = 412 +/- 67 min(-1). For MMP 9/substance P the constants were K-M = 91 +/- 15 mu M and k(cat) = 25 +/- 4 min(-1). Both enzymes cleaved substance P between Gln(6) and Phe(7) and between Gly(9) and Leu(10). 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 mu M 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.