Characterization of GTP-gamma-S binding to isolated human platelet plasma membranes and its relationship with the stimulation of a phospholipase C activity.
Binding parameters for the interaction of GTP-gamma-[35S] with isolated platelet plasma membranes have been studied. Analysis of the data by a non-linear curve fitting program indicates that the interaction can be satisfactory described by a model with a single, high affinity binding site (Kd = 0.3 +/- 0.07 microM and Bm = 0.4 +/- 0.2 nmoles of GTP-gamma-S/mg of membrane protein). Binding is selectively inhibited by GDP-beta-S and GMP-PNP (1 microM), but not affected by ATP, CTP, ITP, or UTP, even at mM concentration. Optimal conditions for the interaction were 30 degrees C and pH 8.0. Incubation of the isolated membranes with GTP-gamma-S results in a measurable phospholipase C activity (as detected both by a breakdown of phosphoinositides and an increase of inositide phosphates) which under our experimental conditions is only slightly enhanced by addition of cytosolic proteins. Our results indicate that platelet plasma membranes contain all the necessary elements for signal transduction through the diacylglycerol/inositolphosphates pathway.
Apitz-Castro
R
R
U-150 INSERM, UA 334 CNRS, Hôpital Lariboisière, Paris, France.
Corvazier
E
E
Jorquera
A
A
Rendu
F
F
Enouf
J
J
Levy-Toledano
S
S
D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D010716:Phosphatidylinositols / Q000097:blood; D015398:Signal Transduction; D013696:Temperature; D013873:Thionucleotides / Q000097:blood*; D013997:Time Factors; D010738:Type C Phospholipases / Q000097:blood*
D010716:Phosphatidylinositols; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D010738:Type C Phospholipases