Real-time in vitro measurement of intrinsic and Ras GAP-mediated GTP hydrolysis.
Ras proteins are small GTPases that exhibit high-affinity binding to GDP and GTP and hydrolyze bound GTP to GDP. The intrinsic GTPase activity of Ras proteins is accelerated by GTPase activating proteins (GAPs), which act to attenuate GTPase signaling by accelerating the conversion of bound GTP to bound GDP. Tumor-associated Ras proteins harbor single amino acid substitutions at residues Gly-12 and Gln-61 that impair the intrinsic and GAP-stimulated GTPase activity, thus rendering these mutant Ras proteins persistently GTP bound and active in the absence of extracellular stimuli. The measurement of GTP hydrolysis in vitro can provide information on the intrinsic activity of, as well as help define, the GAP specificity. Current methods to measure GTP hydrolysis in vitro use either radioactivity-based filter binding assays or measurements of GDP:GTP:P(i) ratios by high-performance liquid chromatography (HPLC). Both provide only endpoint information on the GTP-bound state, can be prone to experimental errors, and do not provide a real-time observation of GTP hydrolysis. The method we describe here uses a fluorescently labeled, phosphate-binding protein (PBP) sensor. A change of protein conformation, caused by binding to a single P(i), is coupled to a measurable increase in fluorescence of the fluorophore. Therefore, this method does allow for real-time monitoring of GTPase activity. This chapter describes the preparation and labeling of the PBP with the MDCC fluorophore and its subsequent use in the measurement of GAP-stimulated GTPase activity. We have used the Ras family small GTPase R-Ras and the GAP-related domain from neurofibromin to demonstrate the application of these protocols.
Shutes
Adam
A
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Der
Channing J
CJ
D003374:Coumarins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D028044:Phosphate-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D003374:Coumarins; D028044:Phosphate-Binding Proteins; D020703:ras GTPase-Activating Proteins; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D018631:ras Proteins