A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.
An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-32P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras(val12)-transformed derivatives. After a 3-hr incubation at 37 degrees C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [alpha-32P]GTP binding and conversion to [alpha-32P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [32P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.
Brownell
H L
HL
Department of Microbiology, Queen's University, Kingston, Ontario, Canada.
Firth
K L
KL
Kawauchi
K
K
Delovitch
T
T
Raptis
L
L
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D018274:Electroporation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D015689:Oncogene Protein p21(ras)