DYNAMIC FEATURES OF PROTHROMBIN INTERACTION WITH PHOSPHOLIPID-VESICLES OF DIFFERENT SIZE AND COMPOSITION - IMPLICATIONS FOR PROTEIN-MEMBRANE CONTACT

The dynamics of prothrombin interaction with membrane vesicles of diff erent size and composition was investigated to ascertain the impact of membrane surface characteristics and particle size on this interactio n. Dissociation rates were highly sensitive to membrane composition an d varied from about 20/s for membranes of 10% PS to 0.1/s for membrane s of 50% PS. Overall affinity also varied by more than two orders of m agnitude. Very small differences between prothrombin binding to SUV ve rsus LUV were found. Association with large unilamellar vesicles (LUV of 115 nm diameter) was about 4-fold slower, when expressed on the bas is of binding sites, than association with small unilamellar vesicles (SUV, 30 nm diameter) of the same composition. Both reactions proceede d at less than 25% of the collisional limit so that the differences we re largely due to intrinsic binding properties. Vesicles of 325 nm dia meter showed even slower association velocities. Dissociation rates fr om LUV were about 2-fold slower than from SUV. Again, these difference s arose primarily from intrinsic binding properties. Dissociation conf ormed to a single first order reaction over a wide range of protein oc cupancy on the membrane. At very high packing density, the dissociatio n rate increased by about 2-fold. At equilibrium, prothrombin preferre d binding to SUV over LUV by about 2-fold. This very small difference, despite substantial differences in phospholipid headgroup packing and hydrocarbon exposure, appeared inconsistent with an important role fo r protein insertion into the hydrocarbon region of the membrane. Howev er, prothrombin-membrane interaction may arise from a series of intera ction forces that have compensating features at equilibrium. The small differences in prothrombin binding to SUV versus LUV, together with d ifferences in the number of protein binding sites per vesicle, were im portant to identify mechanisms of substrate delivery to the active sit e of the prothrombinase enzyme [Lu, Y., & Nelsestuen, G. L. (1996) Bio chemistry 35, 8201-8209].