SOM's antiproliferative effect did not occur in the presence of the nonhydrolyzable guanine nucleotide analog GDP-β-S and was abolished by pretreatment with PTX, implying involvement of G proteins (27).
To determine whether G-proteins are responsible for the dexmedetomidine-induced current, GDP-β-S (2 m m), a non-hydrolysable analog of GDP that competitively inhibits G-proteins, was used in the pipette solution to prevent the postsynaptic activation of α2-adrenoceptors.
Fitting the experimental data to a theoretical model gave us a precise estimate of the longitudinal binding energie (Elong) of GMPCPP (a very slow hydrolysable GTP analog) and GDP tubulin-dimers.
Therefore, the nucleotide dependence of NMM IIA and actin recruitment was investigated in presence of GDPγS, a metabolically inert GDP analog.
Furthermore, intracellular dialysis of 0.5 mM GDP-β-S, a hydrolysis-resistant GDP analog which blocked G-protein activation, was also found to produce no significant effect on SKF83959-induced depolarizing response (GDP-β-S +SKF83959: 6.1±1.4 mV, n = 6; SKF83959 alone: 6.9±1.4 mV, n = 6, unpaired t test, P>0.05).
To test whether a G-protein-mediated pathway is involved, we diffused the nonhydrolyzable GDP analog GDPβS (2 mM), a broad spectrum G-protein inhibitor, from the pipette.
Using isothermal titration calorimetry (ITC), the purified N terminus of FMNL2 (FMNL2N) exclusively interacted with Cdc42 associated with the nonhydrolyzable GTP analog GppNHp, but not with GDP-loaded Cdc42 nor with active Rac1.
"Open" state conformations in the presence of either bound nonhydrolyzable GTP substrate analog guanine 5'-imidotriphosphate (GMPPNP) or product GDP have been shown for Ffh-NG [9], [10].
Further, to probe the effect of GTP/GDP binding on ribosome binding, experiments were conducted in the presence of GDP, GTP and GDPNP (non-hydrolysable GTP analog).
From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP.
