title: Affinity purification of GTPase proteins from oat root plasma membranes using biotinylated GTP.
abstract: Biotinylated GTP was synthesized and it was demonstrated that this ligand was bi-functional: it competed with [3H]Gpp(NH)p for binding to membrane proteins and it bound to immobilized avidin. Peripheral plasma membrane proteins were solubilized in a low-salt wash, incubated with GTP-biotin and biotinylated proteins were coupled to an avidin column. Elution with excess biotin yielded 10 polypeptides as seen with a silver stained SDS-PAGE gel. Antisera raised against Ras, a small GTPase, strongly interacted with three proteins with MW of 38, 27 and 25 kDa and also with 6 other proteins. G alpha-common antibodies interacted with proteins of MW = 66 and 38 kDa. This method enables the rapid purification of GTP-binding proteins and opens the possibility to assign a role to specific GTPases in signal transduction pathways.
authors.affiliation: Department of Plant Physiology and Biochemistry, Vrije Universiteit van Amsterdam, The Netherlands.
authors.forename: A H
authors.initials: AH
authors.lastname: de Boer
authors.forename: E
authors.initials: E
authors.lastname: van Hunnik
authors.forename: H A
authors.initials: HA
authors.lastname: Korthout
authors.forename: N J
authors.initials: NJ
authors.lastname: Sedee
authors.forename: M
authors.initials: M
authors.lastname: Wang
mesh_terms: D001667:Binding, Competitive; D001710:Biotin; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002523:Edible Grain; D020558:GTP Phosphohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006868:Hydrolysis; D010944:Plants / Q000201:enzymology*; D015398:Signal Transduction; D012996:Solutions
chemical_list: D012996:Solutions; D006165:Guanylyl Imidodiphosphate; D001710:Biotin; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases