Influence of anti-tubulin antibodies on muscarinic receptor modulation of G protein GTPase activity in rat striatum.
To understand the role of tubulin, an integral component of neural membranes, in signal transduction processes, the influence of anti-tubulin antibodies on the low Km GTPase activity associated with transducer G proteins was examined in rat striatum. Membranes were prepared from striatum by conventional procedures, and the low Km GTPase activity (EC 3.6.1.-) was determined using [gamma-32P]GTP at 37 degrees in an ATP-regenerating buffer containing 0.2 to 2.0 microM unlabeled GTP. GTPase activity was linear for up to 30 min and was directly proportional to protein concentration. Polyclonal anti-tubulin antibodies, anti-alpha-tubulin antibodies, and anti-beta-tubulin antibodies (10 micrograms) stimulated G protein GTPase activity. Anti-beta-tubulin antibody (10 micrograms) stimulated GTPase activity by about 60% at each time point, while 10 micrograms of either anti-alpha-tubulin or polyclonal anti-tubulin antibodies stimulated GTPase activity by only 20-30% at each time point. The Vmax/Km ratio, an index of the enzyme-substrate interaction, increased by only 26% with the anti-alpha-tubulin antibody and by 52% with anti-beta-tubulin antibody; polyclonal anti-tubulin antibodies did not affect this ratio. GTPase activity was stimulated by acetylcholine in an atropine-sensitive manner. At 100 microM, acetylcholine stimulated GTPase activity by about 50%. Polyclonal anti-tubulin, anti-alpha-tubulin, or anti-beta-tubulin antibodies (10 micrograms) potentiated acetylcholine stimulation of GTPase activity. Two possible mechanisms by which anti-tubulin antibodies could stimulate low Km GTPase activity and potentiate the stimulatory effects of acetylcholine are: (1) by inhibiting GTP binding to beta-tubulin, and (2) by eliminating a chronic inhibitory effect of tubulin on G protein or receptor-G protein interaction.
Ravindra
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Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta 30912.
Aronstam
R S
RS
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001285:Atropine / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000502:physiology*; D014404:Tubulin / Q000276:immunology / Q000502:physiology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D011976:Receptors, Muscarinic; D014404:Tubulin; D001285:Atropine; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000109:Acetylcholine