PHOSPHATIDYLSERINE-CONTAINING MEMBRANES ALTER THE THERMAL-STABILITY OF PROTHROMBINS CATALYTIC DOMAIN - A DIFFERENTIAL SCANNING CALORIMETRIC STUDY

Denaturation profiles of bovine prothrombin and its isolated fragments were examined in the presence of Na(2)EDTA, 5 mM CaCl2, and CaCl2 plus membranes containing l-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with bovine brain phosphatidylserine (PS). We have shown previously [Lentz, B. R., Wu, J. R., Sorrentino, A. M., and Carleton, J. A. (1991) Biophys. J. 60, 70] that binding to PS/POPC (25/75) large unilamellar vesicles resulted in an enthalpy loss in the main endotherm of prothrombin denaturation (T-m similar to 57-58 degrees C) and a comparable enthalpy gain in a minor endotherm (T-m similar to 59 degrees C) accompanying an upward shift in peak temperature (T-m similar to 73 degrees C). This minor endotherm was also responsive to Ca2+ binding and, in the absence of PS/POPC membranes, corresponded to melting of the N-terminal, Ca2+ and membrane binding domain (fragment 1). Peak deconvolution analysis of the prothrombin denaturation profile and extensive studies of the denaturation of isolated prothrombin domains in the presence and absence of PS/POPC vesicles suggested that membrane binding induced changes in the C-terminal catalytic domain of prothrombin (prethrombin 2) and in a domain that links fragment 1 with the catalytic domain (fragment 2). Specifically, the results have confirmed that the fragment 2 domain interacts with and stabilizes the prethrombin 2 domain and also have shown that fragment 2 interacts directly with the membrane. In addition, the results have demonstrated a heretofore unrecognized interaction between the catalytic and membrane binding domains. This interaction can account for another portion of the denaturation enthalpy that appears at high temperatures in the presence of membranes. Our data lead to the hypothesis that membrane-specific alterations in interdomain interactions and domain stability may account for the observation that the C-terminal catalytic domain of prothrombin is more effectively proteolyzed to thrombin on PS-containing membranes as compared to other membrane surfaces [Pei, G., Powers, D. D., Lentz, B. R. (1993) J. Biol. Chem. 268, 3226].