GENERATION OF ANTIBODIES SPECIFIC FOR THE RALA AND RALB GTP-BINDING PROTEINS AND DETERMINATION OF THEIR CONCENTRATION AND DISTRIBUTION IN HUMAN PLATELETS

Peptide specific polyclonal antibodies directed against C-termini of r as p21 related GTP-binding proteins, ralA and ralB, were generated. To assess antibody specificity, cDNAs coding for full length ralA and ra lB were expressed in Escherichia coli as GST fusion proteins. Western blotting analysis using enhanced chemiluminescence technique confirmed that ralA and ralB antibodies were specific for their respective prot ein. To determine the concentration and distribution, varying amounts of GST-ralA and GST-ralB and, human platelet particulate and cytosolic proteins were loaded during Western blotting. The amount of ralA and ralB proteins in the platelet particulate fraction was determined to b e 0.16 +/- 0.017 mu g/mg protein (n = 3) and 0.15 +/- 0.009 mu g/mg pr otein (n = 3) respectively. In the cytosol, only ralB protein was dete cted and its concentration was estimated to be 0.03 +/- 0.009 mu g/mg protein (n = 3). Both ralA and ralB proteins were isoprenylated in the presence of [H-3]mevalonolactone plus rabbit reticulocyte lysate alth ough radioactivity incorporated into ralA was three times higher than that associated with the ralB protein. Addition of geranylgeranyl pyro phosphate to the reaction mixture inhibited incorporation of radioacti vity into ralA and ralB but not cH-ras suggesting that both ralA and r alB proteins are geranylgeranylated. Differential distribution of ralA and ralB GTP-binding proteins in human platelets suggests a distinct role for each of these proteins in platelet function.