Presence of guanine nucleotide-binding proteins in Catharanthus roseus transformed roots

Biochemical analysis revealed the presence of GTP-binding proteins (G-prote ins) in Catharanthus roseus hairy root cultures, In a microsomal fraction, several proteins, with molecular masses of 17, 21, 38, 42, 65, and 79 kDa w ere substrates for ADP-ribosylation by cholera toxin. Antisera raised again st a conserved amino-acid sequence (GTSNSGK-STIVKQMK) of mammalian G alpha subunits recognized three proteins of 42, 50, and 79 kDa, Incubation of nit rocellulose blots with [alpha-P-32]-GTP also indicated the presence of seve ral proteins (17, 21, 50, and 79 kDa) that could bind GTP. In this system, we previously identified a phosphatidylinositol 4,5-bisphosphate-phospholip ase C (PLC, EC 3.1.4.11) activity. As the activation of PLC by C-proteins w as described, we decided to see whether, in our system, G-protein activator s, such as guanosine 5-o-(3-thiotriphosphate) (GTP gamma S) and sodium fluo ride ions, were able to regulate PLC activity in C, roseus transformed root s. Our results show that these agents regulated PLC activity in an inhibito ry fashion and that this effect is dose-dependent. GTP was ineffective in p roducing either stimulation or inhibition of PLC activity, Our results demo nstrate that non-hydrolyzable guanine nucleotides acid fluoride ions exert an inhibitory effect on membrane PLC activity. In summary, a set of protein s of 17, 21, 38, 42, 50, and 79 kDa present in C. roseus transformed roots possessed at least two of the three main characteristics of a CTP-binding p rotein, and one of these proteins may be involved in the regulation of PLC activity in C. roseus transformed roots.