Immunodetection of ralA and ralB GTP-binding proteins in various rat tissues and platelets.
Polyclonal antibodies were generated against a synthetic peptide corresponding to the C-terminal (amino acids 192-204) region of ralA and ralB GTP-binding proteins. The ralA and ralB antibodies recognized a 27 kDa protein in the human platelet particulate fraction. Incubation of ralA antibodies with ralB immunizing peptide and ralB antibodies with ralA immunizing peptide prior to Western blotting did not abolish the ability of antibodies to recognize the 27 kDa protein in human platelet particulate fraction. However, when antibodies were incubated with the respective immunizing peptide prior to Western blotting, the 27 kDa human platelet protein was no longer recognized by the antibodies. Incubation of nitrocellulose blots containing polypeptides separated using SDS-PAGE with [alpha-32P]GTP demonstrated the presence of GTP-binding proteins of molecular mass between 23-27 kDa in rat platelets and the various tissues tested. Analysis using subtype specific antibodies demonstrated that both ralA and ralB GTP-binding proteins were expressed in rat platelets and the various tissues tested. The protein recognized by the ralA and ralB antibodies in rat tissues and platelets had mobility on SDS-PAGE identical to that of the human platelet ral protein. Varying amounts of these proteins were detected in all the tissues tested except white muscle which contained very low level of ralB protein. The widespread distribution of ralA and ralB GTP-binding proteins suggests that they may participate in a common pathway in mammalian cells and tissues.
Bhullar
R P
RP
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Yang
S
S
D000818:Animals; D001792:Blood Platelets / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000276:immunology; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D010446:Peptide Fragments / Q000276:immunology; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D051381:Rats; D020725:ral GTP-Binding Proteins
D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins