Improved methods to detect GTP-binding proteins from plants

Abstract

Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of mossFunaria hygrometrica and coleoptiles of corn (Zea mays) and sorghum(Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer polyclonal anti-Ga (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α -subunit of heterotrimeric G-proteins (M_r 41-43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Ga (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparent K_m of the NAD has been determined to be approximately 1.5µ M for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2-27-fold. One to three polypeptides representing the α -subunit of heterotrimeric G-proteins of (Mr 37-43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.