title: GTP-specific fab fragment-based GTPase activity assay.
abstract: GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.
authors.affiliation: †Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, Third Floor, FI-20520 Turku, Finland.
authors.forename: Kari
authors.initials: K
authors.lastname: Kopra
authors.forename: Anita
authors.initials: A
authors.lastname: Rozwandowicz-Jansen
authors.forename: Markku
authors.initials: M
authors.lastname: Syrjänpää
authors.affiliation: ∥Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.
authors.forename: Olga
authors.initials: O
authors.lastname: Blaževitš
authors.affiliation: ∥Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.
authors.forename: Alessio
authors.initials: A
authors.lastname: Ligabue
authors.affiliation: ⊥University Hospital Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Veltel
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
authors.affiliation: ∥Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Abankwa
authors.affiliation: †Institute of Biomedicine, Department of Cell Biology and Anatomy, University of Turku, Kiinamyllynkatu 10, Third Floor, FI-20520 Turku, Finland.
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D000918:Antibody Specificity; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*
chemical_list: D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D007140:Immunoglobulin Fab Fragments; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases