Effects of water soluble phosphotidylserine on bovine factor Xa: functional and structural changes plus dimerization.
Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor X(a). Activity measurements along with the fluorescence of active-site-labeled human factor X(a) 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 X(a) conformational change. The purpose of this paper is to extend these observations to bovine factor X(a) and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor X(a) 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 X(a). C6PS induced a 70-fold increase in bovine factor X(a)'s autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor X(a). In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor X(a) induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca(2+)-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 X(a) elicits different structural and functional responses.
Majumder
Rinku
R
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260, USA.
Wang
Jianfang
J
Lentz
Barry R
BR
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003619:Dansyl Compounds; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015951:Factor Xa / Q000737:chemistry*; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012995:Solubility; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry*
D003619:Dansyl Compounds; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010718:Phosphatidylserines; D012996:Solutions; D014867:Water; D015951:Factor Xa