Isolation and purification of a small-molecular-weight GTP-binding protein from plants

As part of testing the hypothesis that signal transduction in higher plants is mediated by G-protein(s), a 24-kDa GTP-binding protein was isolated and purified to near homogeneity from 3.5-day-old dark grown oat (Avena) seedlings by hydroxyapatite, GTP-agarose, and Sephacryl S-200 chromatographies. The protein bound maximally 0.9 +/- 0.1 mol of [35S]guanosine 5'-(3-O-thio) triphosphate (GTP gamma S)/mol of protein with an apparent Kd of approximately 32 nM. Although binding of [35S]GTP gamma S was inhibited by both GTP and GDP, no inhibition of [35S]GTP gamma S binding was observed in the presence of ATP, UTP, CTP, ITP, or TTP. The 24-kDa protein hydrolyzed GTP with a rate of 0.06 mol Pi/mol protein/min. The results suggest that this GTP-binding protein is a unique plant GTP-binding protein that exhibits characteristics similar to those of animal ras-related GTP-binding proteins.