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Quech-flow kinetic measurement of individual reactions of G-protein-catalyzed GTPase cycle

Elsevier Science & Technology
DOI: 10.1016/s0076-6879(02)44727-1
  • Section Iii. Functional Analysis Of G Protein Subunits
  • Biology
  • Chemistry


Publisher Summary Fast and efficient G-protein-mediated signaling depends on the kinetic balance of the individual reactions of the GTPase cycle: GTP binding, hydrolysis of bound GTP, and dissociation of GDP. Each step of the GTPase cycle is determined both by the instrinsic activity of the Gα subunit and by the combined regulatory inputs of Gβγ, agonist-liganded receptors, and GTPase-activating proteins (GAPs). Quench-flow kinetic assays use a mechanical mixing device to initiate fast reactions of interest, incubate them for set times, and then quench the reaction by addition of another reagent, all on the time scale of 1-10,000 ms. After final quenching, the reaction mixture is expelled to allow the assay of product or of remaining substrate. Quench-flow assays are generally considered less efficient than stopped-flow spectrophotometry because stopped flow provides a continuous reaction trace whereas quench flow requires a separate assay for each time point. However, quench flow is applicable when optical reporters are unavailable. Quench flow also provides absolute chemical quantitation of the reaction, which is often impossible with stopped flow.

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