Evidence from total internal reflection fluorescence microscopy for calcium-independent binding of prothrombin to negatively charged planar phospholipid membranes.
Measurements to test for a proposed Ca2+-independent interaction of prothrombin with membranes containing acidic phospholipids are described. Fluorescein-labeled bovine prothrombin and its amino- and carboxy-terminal peptides, prothrombin fragment 1 and prethrombin 1, were added at various concentrations in the presence or absence of Ca2+ to the aqueous space bathing substrate-supported planar membranes composed of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC), POPC/bovine brain phosphatidylserine (bovPS) (70:30 mol/mol), or POPC/1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) (70:30 mol/mol). Total internal reflection fluorescence microscopy (TIRFM) at the membrane-solution interface showed a significant enhancement by acidic lipids of prothrombin and prothrombin fragment 1 binding in the presence of 5 mM Ca2+, with apparent dissociation constants of 0.4 and 1 microM, respectively. TIRFM measurements indicated that bovPS and DOPG also significantly enhanced the binding of fluorescein-labeled prothrombin to the planar membranes in the absence of Ca2+, with apparent dissociation constants (13-30 microM) at least an order of magnitude larger than the Ca(2+)-dependent constant for prothrombin binding. Association of prethrombin 1 but not prothrombin fragment 1 with membranes in the absence of Ca2+ was enhanced by the presence of bovPS in the membranes, which suggests that the Ca(2+)-independent binding site(s) is (are) in the prethrombin 1 but not the fragment 1 portion of prothrombin.
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
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Tendian
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Lentz
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Thompson
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D004058:Diffusion; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D008081:Liposomes; D008856:Microscopy, Fluorescence / Q000379:methods; D010743:Phospholipids / Q000378:metabolism*; D011516:Prothrombin / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship
D008081:Liposomes; D010743:Phospholipids; D011516:Prothrombin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium