Solubilization and characterization of a platelet membrane ADP-binding protein.
Previous studies have shown that platelet membranes bind radiolabeled ADP and have nucleoside diphosphokinase activity which transforms added ADP to ATP. In order to further characterize these reactions, the ADP-binding and nucleoside diphosphokinase activity of purified platelet membranes were solubilized by freeze-thaw injury followed by extraction with isotonic buffered saline. Up to 80% of membrane ADP-binding activity was solubilized along with 20% of the total membrane protein, a 4-fold purification. A Millipore filter binding assay was developed to detect the soluble binding protein using [3H]ADP as radioligand. Binding of [3H]ADP was rapid, reversible, saturable, and was destroyed by heat, trypsin digestion, and 1 mM N-ethylmaleimide. By Scatchard analysis, there was a single class of binding sites with a Kd of 3.8 x 10(-7) M. Unlabeled nucleotides competed with [3H]ADP with the following potency series: ATP = ADP greater than AMP greater than adenosine. The solubilized nucleoside diphosphokinase activity could be separated from ADP-binding activity by ultracentrifugation on 5 to 20% sucrose density gradients containing 0.6 M KCl suggesting that the activities reside on separate molecules. Hydrodynamic parameters were calculated for the binding protein by gel filtration and ultracentrifugation. The s20,w was 4.1, Stoke's radius 35 x 10(-8)cm, axial ratio (f/fo) 1.09, and the Mr = 61,000. The studies suggest that this platelet ADP-binding protein may act as the receptor for initiating ADP-induced aggregation and release.
D000244:Adenosine Diphosphate / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002462:Cell Membrane / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D011092:Polyethylene Glycols; D011487:Protein Conformation
D002352:Carrier Proteins; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D000244:Adenosine Diphosphate