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Characterization of Low Km GTPase Activity in Rat Brain: Comparison of Opioid and Muscarinic Receptor Stimulation

1988; Elsevier BV; Volume: 77; Issue: 4 Linguagem: Inglês

10.1002/jps.2600770415

1520-6017

Wayne Hoss, Paul Franklin, Shahnaz Ghodsi-Hovsepian,

Receptor Mechanisms and Signaling

Guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins) have an essential role in mediating the actions of drugs on neurotransmitter receptors by coupling them to their effectors with the attendant hydrolysis of GTP. The resulting GTPase activity was characterized in rat brain with a view toward selecting conditions under which specific hormone-stimulated activity could be monitored. Kinetic analysis with washed membranes suggested the presence of two distinct GTPases, a low Km GTPase with an apparent Km value of 0.35 ± 0.04 μM and apparent Vmax of 108 pmol min–1 mg protein–1, together with a much higher Km component. Low Km (but not high Km) GTPase activity is stimulated by muscarinic and opioid agonists and inhibited by a nonhydrolyzable analogue of GTP, providing further evidence that the low Km component is a distinct enzyme. The activity of the low Km component is a linear function of protein concentration (20–100 μg/mL), time (2–10 min), and temperature (25–37 C). The specific activity of the low Km component is selectively increased by ~50% in purified synaptic membranes compared with the washed membrane preparation. Both carbamylcholine-stimulated and basal low Km GTPase activities, but not the high Km component, are inhibited by a nonhydrolyzable analogue of GTP but not by the comparable analogue of ATP, demonstrating the specificity of low Km GTPase for guanine nucleotides. Opioid- and muscarinic-stimulated GTPase activities are additive in brain, suggesting that the two receptor systems are associated with different domains of G-proteins. It is concluded that specific hormone-stimulated GTPase activities can be measured in brain under appropriate conditions including submicromolar GTP concentrations, the presence of a nucleotide regenerating system, and suppression of ATPases. The results indicate that hormone-stimulated low Km GTPase can serve as an independent measure of drug–receptor interactions in brain.