Affinity purification of GTPase proteins from oat root plasma membranes using biotinylated GTP

FEBS Letters

de Boer, Albertus H.²  Korthout, Henrie A.A.J.²  Sedee, Norbert J.A.¹  van Hunnik, Eddy²  Wang, Mei¹ 

[1] Center for Phytotechnology, RULITNO, Department of Plant Molecular Biotechnology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands;Department of Plant Physiology and Biochemistry, Institute for Molecular Biological Sciences, BioCentrum, Vrije Universiteit van Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands

关键词: G-protein;    GTPase;    GTP-biotin;    Monomeric avidin;    Plant root;    Plasma membrane;    Avena sativa;    EDC;    1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;    MEGA 9;    nonoyl-N-methylglucamide;    PM;    plasma membrane;   

DOI  :  10.1016/0014-5793(94)80209-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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_α-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.

【 授权许可】

Unknown