title: High-throughput assay for profiling the substrate specificity of Rab GTPase-activating proteins.
abstract: Measurement of intrinsic as well as GTPase-Activating Protein (GAP)-catalyzed GTP hydrolysis is central to understanding the molecular mechanism and function of GTPases in diverse cellular processes. For the Rab GTPase family, which comprises at least 60 distinct proteins in humans, putative GAPs have been identified from both eukaryotic organisms and pathogenic bacteria. A major obstacle has involved identification of target substrates and determination of the specificity for the Rab family. Here, we describe a sensitive, high-throughput method to quantitatively profile GAP activity for Rab GTPases in microplate format based on detection of inorganic phosphate released after GTP hydrolysis. The method takes advantage of a well-characterized fluorescent phosphate sensor, requires relatively low protein concentrations, and can in principle be applied to any GAP-GTPase system.
authors: Department of Biochemistry and Molecular Pharmacology, Program in Molecular Medicine, University of Massachusetts Medical School, Two Biotech, Suite 119, 373 Plantation Street, Worcester, MA, 01605, USA, ashwini.mishra@umassmed.edu.
authors: Ashwini K
authors: AK
authors: Mishra
authors: David G
authors: DG
authors: Lambright
mesh_terms: D055162:Biocatalysis; D029968:Escherichia coli Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006868:Hydrolysis; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D020691:rab GTP-Binding Proteins