The effects of hexanol on Galpha(i) subunits of heterotrimeric G proteins.
Alcohols and other anesthetics interfere with the function of a variety of systems regulated by guanosine triphosphate (GTP)-binding proteins (G proteins). We examined the effect of hexanol on the activity of the alpha subunit (Galpha(i1)) of heterotrimeric G proteins. The GTP hydrolysis activity of recombinant Galpha(i1) was 0.029 mole Pi. mole Galpha(i1)(-1) x min(-1) and was inhibited by hexanol at concentrations larger than 10 mM, with a 50% inhibitory concentration of 22 mM. Circular dichroism spectroscopy revealed that hexanol decreased the denaturation temperature of Galpha(i1) from 47.2 degrees C to 42.5 degrees C without altering its secondary structure at 10 degrees C. Hexanol (30 mM) reduced the amount of monomeric Galpha(i1) in solution measured by size-exclusion chromatography, indicating that hexanol caused protein aggregation. However, the rate of GTPgammaS binding to Galpha(i) immunoprecipitated from airway smooth muscle membranes was not affected by 30 mM hexanol. Excluding the apparent inhibition of recombinant Galpha(i1) resulting from aggregation-induced artifact, we found no evidence that the hexanol-induced inhibition of receptor-activated Galpha(i)-coupled pathways in intact airway smooth muscle resulted from direct inhibition of the intrinsic rate of [(35)S]GTPgammaS binding to Galpha(i).


CONCLUSIONS
Although the alpha subunit of heterotrimeric G proteins is a potential target of anesthetics, we found no evidence that hexanol affects the ability of the Galpha(i) subunit to bind or hydrolyze guanosine triphosphate, either in purified subunits or in subunits derived from smooth muscle cell membranes. This finding implies that this is not a mechanism by which hexanol interferes with receptor-G protein function.
Departments of Anesthesiology and. Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
John
J
Streiff
David O
DO
Warner
Elena
E
Klimtchuk
William J
WJ
Perkins
Kristofer
K
Jones
Keith A
KA
Jones
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000187:drug effects*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D000441:Hexanols / Q000494:pharmacology*; D006358:Hot Temperature; D066298:In Vitro Techniques; D009130:Muscle, Smooth / Q000187:drug effects; D011233:Precipitin Tests; D011489:Protein Denaturation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry; D013552:Swine
D000441:Hexanols; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000251:Adenosine Triphosphatases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go