Generation of antibodies specific for the RalA and RalB GTP-binding proteins and determination of their concentration and distribution in human platelets

Abstract

Peptide specific polyclonal antibodies directed against C-termini of ras p21 related GTP-binding proteins, ralA and ralB, were generated. To assess antibody specificity, cDNAs coding for full length ralA and ralB 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 protein. 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 be 0.16±0.017 μg/mg protein ( n = 3) and 0.15 ± 0.009 μg/mg protein ( n = 3) respectively. In the cytosol, only ralB protein was detected and its concentration was estimated to be 0.03±0.009 μg/mg protein ( n = 3). Both ralA and ralB proteins were isoprenylated in the presence of [ 3H]mevalonolactone plus rabbit reticulocyte lysate although radioactivity incorporated into ralA was three times higher than that associated with the ralB protein. Addition of geranylgeranyl pyrophosphate to the reaction mixture inhibited incorporation of radioactivity into ralA and ralB but not cH-ras suggesting that both ralA and ralB 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.