Calcium and prothrombin changed the observed phase transition temperature (Tm) of appropriately constituted phospholipid vesicles. The results are consistent with clustering of the acidic phospholipid molecules to form a prothrombin binding site. Transition temperatures were obtained by monitoring the fluorescence intensity and fluorescence depolarization of the lipid-soluble probes 8-anilino-1-naphthalenesulfonic acid and diphenylhexatriene, respectively. In all cases, calcium and prothrombin caused the observed Tm to increase or decrease toward the value of the pure neutral phospholipid. For example, calcium caused a 2°C increase in the observed Tm of vesicles containing 20% bovine brain phosphatidylserine (PS) and 80% dipalmitoylphosphatidylcholine (DPPC). Prothrombin binding to these vesicles caused a further 2°C increase in the Tm. Conversely, calcium or calcium plus prothrombin caused 2 and 3.5°C decreases, respectively, in the observed Tm of membranes of 30% dipalmitoylphosphatidic acid and 70% dimyristoylphosphatidylcholine. The changes are due to clustering of the acidic phospholipid molecules, which enriches the bulk membrane in the neutral phospholipid. The observed Tm therefore increases or decreases, depending on whether the acidic phospholipid component had a Tm higher or lower than the neutral phospholipid. In agreement with this interpretation and with previous studies, manganese caused negligible changes in the Tm and inhibited the effect of calcium. In addition, the concentration dependence of the calcium-induced increase in the Tm (for PS-DPPC vesicles) indicated a dissociation constant of about 0.5 mM for calcium-phosphatidylserine binding, which agrees with direct calcium binding measurements. The inhibitory effect of manganese on the calcium-induced changes was overcome by prothrombin; in the presence of manganese and calcium, prothrombin still induced the full 4°C increase. Light-scattering studies demonstrated that, in both cases, prothrombin-induced changes in the Tm correlate closely with actual prothrombin binding to the phospholipid vesicles. Lateral phase separation appears to be an integral part of prothrombin-membrane binding.
|Original language||English (US)|
|Number of pages||7|
|State||Published - 1981|