In vitro study of G protein activation by 35S-GTPγS binding to platelet membrane.: Effects of different modulators

Activation of guanine nucleotide triphosphate (GTP)-binding protein by liganded-receptor was investigated in platelet membrane by measuring alpha 2-adrenergic receptor agonist (the bromoxidine or UK-14304)-induced S-35-GTP gamma S binding in the presence or absence of modulators such as magnesium, sodium chloride and guanine nucleotide diphosphate (GDP). In basal conditions, with millimolar levels of magnesium (5mM), results indicated high spontaneous, non-receptor-mediated S-35-GTP gamma S at 37 degrees C. The addition of 100mM NaCl and 2 mu M GDP reduced the absolute levels of bound radioligand, but increased the effect of the agonist. At low temperature (4 degrees C) the presence of GDP was not required for an agonist effect. If magnesium was reduced to picomolar levels (max 1 mu mol) a S-35-GTP gamma S release occurred in the presence of the alpha 2-AR agonist. These data showed, contrary to some cell systems, that platelet alpha 2-adrenoceptor required the presence of magnesium, guanine nucleotide diphosphate and sodium chloride for activation of G-protein, when the reaction was followed at physiological temperature.