title: Characterization of low Km GTPase activity in rat brain: comparison of opioid and muscarinic receptor stimulation.
abstract: 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 microM 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 micrograms/mL), time (2-10 min), and temperature (25-37 degrees C). The specific activity of the low Km component is selectively increased by approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Medicinal Chemistry, University of Toledo, College of Pharmacy, OH 43606.
authors.forename: W
authors.initials: W
authors.lastname: Hoss
authors.forename: P H
authors.initials: PH
authors.lastname: Franklin
authors.forename: S
authors.initials: S
authors.lastname: Ghodsi-Hovsepian
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011957:Receptors, Opioid / Q000378:metabolism*; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism; D013696:Temperature
chemical_list: D006150:Guanine Nucleotides; D011976:Receptors, Muscarinic; D011957:Receptors, Opioid; D006160:Guanosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases