Evaluation of membrane phase behavior as a tool to detect extrinsic protein-induced domain formation: binding of prothrombin to phosphatidylserine/phosphatidylcholine vesicles.
The temperature-composition phase diagram of mixed dimyristoylphosphatidylserine (DMPS) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles was determined in the presence and absence of bound bovine prothrombin by monitoring the phospholipid order-disorder phase separation using diphenylhexatriene (DPH) fluorescence anisotropy. The shape of the membrane temperature-composition diagram was essentially unaltered by the binding of prothrombin in the presence of Ca2+ although the two-phase (gel/fluid) region was slightly narrowed and shifted by 1-10 degrees C to higher temperatures. This result does not support the popular idea that extensive domains rich in negatively charged phospholipid are induced in response to prothrombin binding. Instead of implying domain formation, our results demonstrate that the observed increase in melting temperature associated with binding of prothrombin to acidic phospholipid membranes can be accounted for by the observed altered membrane order both in the fluid and in the solid lamellar phases. The membrane order in the liquid-crystalline phase increased with increased acidic lipid content, and much more so for DMPS than for dipentadecanoylphosphatidylglycerol (DC15PG). These results demonstrate that simple shifts in membrane phase behavior cannot be properly interpreted to prove the existence of charged lipid domains. In addition, we report the unexpected observation that prothrombin increased the anisotropy of DPH in DMPS/DMPC vesicles in the liquid-crystalline phase in the absence of Ca2+ as well as in its presence. This effect was seen to a lesser extent and only at a much higher charged-lipid content for DC15PG/DMPC vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill 27599-7260.
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Tendian
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Lentz
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism*; D005454:Fluorescence Polarization; D008081:Liposomes / Q000378:metabolism*; D008560:Membrane Fluidity; D010446:Peptide Fragments / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D013696:Temperature
D008081:Liposomes; D010446:Peptide Fragments; D010718:Phosphatidylserines; C042859:dimyristoylphosphatidylserine; D011516:Prothrombin; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine