Abstract
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.
Highlights
The binding interaction of bone Gla protein (BGP), catalyzed by a vitamin K-dependent carboxylase in liver (6, or osteocalcin, to phospholipid vesiclesin thepresence 7)
P C P s inhibition was achieved under conditions used as evidenced complex by systematically varying the concentration of BGP. by the curve becoming saturated at 50% maximum thrombin
82% of pyr-Va was displaced with Factor Xa, prothrombin,andFactor Va, andnota from the vesicles at thehighest BGP concentration (7.5 p ~ ) . consequence of BGP directly interacting with thrombin, Fac-Control experiments involving the addition of BGP into a torXa, Factor Va, or DAPA, several assays using excess cuvette, to a final concentration of 5 p ~co,ntaining only pyr- phospholipid concentrations (30 pM PCPs) were performed
Summary
BGP was added into theassay mixtures employing phospholipid-limiting concentrations (0.5 pM PCPs). Equilibrium gel filtration employingnative, unlabeled, BGP at a concentration used in radiolabeled protein experiments (8p M ) was carried out to determine if the lipid-binding ability of BGP was altered as a result of radioiodination For these experiments it was necessary to increase the column volume from 20 to 40 ml since measurement of absorbance required. Quantitation of bound BGP determined from the trough values, yielded a dissociation constant of 9.7 p ~ an, d an extrapolated protein/vesicle stoichiometry of 144 (24 PCPs lipids/BGP) (data not shown). These values are consistent with the binding parameters determined from peak analysis. Experimentsinvolving BGP concentrations greater than 17 pM (0.1 mg/ml) were not carried out because of the excessive amounts of BGP required
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