Plasma membranes (1-2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 FM [ ( -Y -~~P ] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)regenerating buffer at 37ยฐC for 1 h; during this incubation, the [32P]GTP i s hydrolyzed and the nucleotide that is predominantly bound to the membranes is [32Pl guanosine diphosphate (GDP). [32P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca2+, Mg2+, Mn2+, and Zn2+ maximally inhibited GDP release by 80-90%, whereas, 5 m M Cu2+ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One pM Gpp(NH)p (5'-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [32P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange.
In the rat membranes, 1-100 nM glucagon (used as a positive control) stimulated [32P]GDP release by about 17% (P < .05); similarly, 0.1-100 nM insulin stimulated [32P]GDP release by 10-13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [32P]GDP release by 7-10%. In the rat membranes, 10 nM insulin stimulated
[32P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [32P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 pg of insulin receptor antibody stimulated [32P]GDP release by 12-22% (P < .05) and 7-1476 in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.