Measurement of KRAS-GTPase Activity.

Dana Rabara, Andrew G Stephen

Methods in molecular biology (Clifton, N.J.) 2024

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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. © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.