Measurement of KRAS-GTPase Activity

Dana Rabara; Andrew G Stephen

doi:10.1007/978-1-0716-3822-4_7

Measurement of KRAS-GTPase Activity

Methods Mol Biol. 2024:2797:91-102. doi: 10.1007/978-1-0716-3822-4_7.

Authors

Dana Rabara¹, Andrew G Stephen²

Affiliations

¹ NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA. dana.rabara@nih.gov.
² NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

PMID: 38570454
DOI: 10.1007/978-1-0716-3822-4_7

Abstract

Oncogenic mutations in KRAS typically impact the GAP-mediated and intrinsic GTP hydrolysis activity resulting in elevated levels of cellular KRAS-GTP. The development of biochemical assays for GTPase activity provides an opportunity to quantitatively measure the impact of these mutations on GTP hydrolysis. Here we describe a biochemical assay that measures the release of free phosphate upon hydrolysis of the GTP nucleotide and allows the measurement of intrinsic or GAP-stimulated GTP hydrolysis by KRAS. This assay can be used to measure GTPase activity under single turnover conditions.

Keywords: Free phosphate; GAP; GTPase activity; Kinetic measurement; NF1; Phosphate sensor; RAS; RASA1.

MeSH terms

GTPase-Activating Proteins* / metabolism
Guanosine Triphosphate
Hydrolysis
Kinetics
Mutation
Proto-Oncogene Proteins p21(ras)* / genetics

Substances

Proto-Oncogene Proteins p21(ras)
Guanosine Triphosphate
GTPase-Activating Proteins