DETERMINATION OF ABSOLUTE AMOUNTS OF GDP AND GTP BOUND TO RAS IN MAMMALIAN-CELLS - COMPARISON OF PARENTAL AND RAS-OVERPRODUCING NIH 3T3 FIBROBLASTS

We devised enzyme-based methods to measure fmol amounts of GDP and GTP and applied these methods to measure absolute amounts of Ras-bound GD P and GTP in NIH 3T3 fibroblasts, We found that parental NIH 3T3 cells contained 509 and 1.3 fmol of Ras-bound GDP and GTP, respectively, pe r mg of cellular protein and that stable transfectants of NIH 3T3 cell s overexpressing wild-type Ha-Ras contained 7008 and 21.3 fmol of Ras- bound GDP and GTP, respectively, per mg of cellular protein; thus, in both cell types <0.3% of Ras was in the active GTP-bound state, In con trast, NIH 3T3 cells overexpressing an activated form of Ha-Ras contai ned 5013 and 2049 fmol of Ras-bound GDP and GTP, respectively, per mg of protein, yielding 29% of Ras in the GTP-bound state, Since intracel lular Ras is probably all in a guanine-nucleotide bound state, this me thod allows one to calculate the number of Ras molecules in each cell: parental NIH 3T3 cells and the Ha-Ras overproducing cells contain app roximate to 20,000 and approximate to 275,000 Ras molecules per cell, respectively, We also incubated parental NIH 3T3 cells with (PO4)-P-32 and determined the radioactivity in Ras-bound GDP and GTP and the spe cific activity of cytosolic GDP and GTP; these experiments indicated t hat Ras-bound GTP may not be in equilibrium with the total cytosolic G TP pool.