Effects of Water Soluble Phosphotidylserine on Bovine Factor Xa: Functional and Structural Changes Plus Dimerization |
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Authors: | Rinku Majumder Jianfang Wang Barry R. Lentz |
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Affiliation: | Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260 |
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Abstract: | Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor Xa. Activity measurements along with the fluorescence of active-site-labeled human factor Xa showed that two linked sites specifically regulate the active site conformation and proteolytic activity of the human enzyme. These results imply, but cannot demonstrate, a C6PS-induced factor Xa conformational change. The purpose of this paper is to extend these observations to bovine factor Xa and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor Xa also varied with C6PS concentration in a sigmoidal manner, whereas amidolytic activity of unlabeled enzyme varied in a simple hyperbolic fashion, also as seen for human factor Xa. C6PS induced a 70-fold increase in bovine factor Xa's autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor Xa. In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor Xa induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca2+-dependent dimer formation, as demonstrated by changes in intrinsic fluorescence and quantitative native gel electrophoresis. Dimerization produced further change in secondary structure, either inter- or intramolecularly. These results, along with results presented previously, support a model in which C6PS binds in a roughly sequential fashion to two linked sites whose occupancy in both human and bovine factor Xa elicits different structural and functional responses. |
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Keywords: | PS, Phosphatidylserine C6PC, 1,2-dicaproyl-snglycero- 3-phosphocholine C6PG, 1,2-dicaproyl-sn-glycero-3-phosphorac- 1-glycerol C6PS, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine POPC, 1,2-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine DOPG, 1,2-dioleoylphosphatidylglycerol DAG, Diacylglycerol GPS, Glycerol phosphorylserine DEGR-CK, [5-(dimethylamino)-1-naphthalenesulfonyl] glutamylgycylarginyl chloromethyl ketone DEGR-Xa, Factor Xa modified with DEGRCK S2765, N-2-benzyloxycarbonyl-d-arginyl-glycyl-Larginine-P-nitroanilide- dihydrochloride CD, Circular dichroism SDS-PAGE, Sodium dodecylsulfate polyacrylamide gel electrophoresis |
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