We have investigated the kinetics and mechanism of prothrombin-membrane vesicle interaction by using stopped-flow light scattering. Under conditions of approximately physiological protein concentration (≤3 μM prothrombin), prothrombin interaction with the vesicles was modeled according to a simple bimolecular process with noninteracting prothrombin binding sites on the vesicle. The association rate constant (per protein binding site) for interaction of prothrombin with vesicles containing 20% phosphatidylserine-80% phosphatidylcholine at 10 °C, in buffer containing 3 mM calcium, is (1 ± 0.1) × 107 M-1·s-1. This corresponds to a 10% collision efficiency. The reverse process is a first-order dissociation with a rate constant of 3 ± 1 s-1. Off-rate experiments conducted by sample dilution were consistent with these values. With varying membrane compositions the association process was found to be somewhat cooperative with respect to phosphatidylserine, but dissociation was unaffected by phosphatidylserine density. The activation energy for prothrombin-membrane association varied with the amount of acidic phospholipid in the membrane. Membranes of 10% phosphatidylserine gave an activation energy of about 9 kcal/mol while those of 40% phosphatidylserine gave a value of 4 kcal/mol. For these same membranes the collision efficiency was estimated to be 3 and 20%, respectively. This trend in the activation energy suggests that as the acidic phospholipid content increases, the association energy of activation becomes characteristic of a diffusion-controlled reaction. Dissociation rate constants were obtained by mixing prothrombin-membrane complexes with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). It was found that a necessary population of calcium ions exchanged rapidly from the prothrombin-membrane complex but that this population could be replaced by magnesium or manganese. A second population of essential ions, specific for calcium, exchanged slowly and, under certain conditions, appeared to be released at the rate of prothrombin-membrane dissociation. Under conditions of high free-protein concentrations the association process became complex and had lower rate constants. The anomalous binding characteristics were observed under conditions that are not likely to be physiologically important.
|Original language||English (US)|
|Number of pages||11|
|State||Published - 1982|