Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: comparison of parental and Ras-overproducing NIH 3T3 fibroblasts.

Abstract

We devised enzyme-based methods to measure fmol amounts of GDP and GTP and applied these methods to measure absolute amounts of Ras-bound GDP 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, per mg of cellular protein and that stable transfectants of NIH 3T3 cells 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 contrast, NIH 3T3 cells overexpressing an activated form of Ha-Ras contained 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 intracellular Ras is probably all in a guanine-nucleotide bound state, this method allows one to calculate the number of Ras molecules in each cell: parental NIH 3T3 cells and the Ha-Ras overproducing cells contain approximately 20,000 and approximately 275,000 Ras molecules per cell, respectively. We also incubated parental NIH 3T3 cells with 32PO4 and determined the radioactivity in Ras-bound GDP and GTP and the specific activity of cytosolic GDP and GTP; these experiments indicated that Ras-bound GTP may not be in equilibrium with the total cytosolic GTP pool.