Affinity purification of GTPase proteins from oat root plasma membranes using biotinylated GTP.
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.
de Boer
A H
AH
Department of Plant Physiology and Biochemistry, Vrije Universiteit van Amsterdam, The Netherlands.
van Hunnik
E
E
Korthout
H A
HA
Sedee
N J
NJ
Wang
M
M
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
D012996:Solutions; D006165:Guanylyl Imidodiphosphate; D001710:Biotin; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases