The interaction of thrombomodulin with Ca2+ - Wiley Online Library

11 downloads 0 Views 1MB Size Report
Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888±2892]. ...... bovine [31] and hamster (Q. Chou, W. H. Andrews and D. Light,.
Eur. J. Biochem. 262, 522±533 (1999) q FEBS 1999

The interaction of thrombomodulin with Ca2+ David R. Light1, Charles B. Glaser2, Melissa Betts2, Eric Blasko2, Elizabeth Campbell3, Jeffrey H. Clarke2, Michael McCaman3, Kirk McLean1, Mariko Nagashima1, John F. Parkinson4, Galina Rumennik1, Tish Young2 and John Morser1 1

Cardiovascular Research, 2Biological Research, 3Process Development and 4Immunology Research, Berlex Biosciences, Richmond, CA, USA

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888±2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4±Pro490), TME1±6 (Cys227±Cys462) and TMEi4±6 (Val345± Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd