The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles.
The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.
Gendreau
M A
MA
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405-0068.
Krishnaswamy
S
S
Mann
K G
KG
D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D005165:Factor V / Q000378:metabolism; D015943:Factor Va; D066298:In Vitro Techniques; D008051:Lipid Bilayers; D008970:Molecular Weight; D015675:Osteocalcin; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011516:Prothrombin / Q000378:metabolism; D013816:Thermodynamics
D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D010743:Phospholipids; D015675:Osteocalcin; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin