Effect of halothane on the guanosine 5' triphosphate binding activity of G-protein alphai subunits.
Effect of halothane on the guanosine 5' triphosphate binding activity of G-protein alphai subunits.
复制标题
氟烷对 G 蛋白 αi 亚基的鸟苷 5 三磷酸结合活性的影响。
DOI：
10.1097/00000542-200307000-00019
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发表时间：
2003
期刊：
影响因子：
8.8
通讯作者：
Jones,KeithA
中科院分区：
文献类型：
--
作者：
Streiff,John;Jones,Kristofer;Perkins,WilliamJ;Warner,DavidO;Jones,KeithA
受体介导的气道平滑肌产生的力的增加被麻醉剂如氟烷减弱。已知鸟苷5 '-三磷酸（GTP）结合蛋白α亚单位（Galpha（i））参与气道平滑肌中的力的调节。作者假设氟烷会抑制G α（i）亚单位结合GTP的非水解类似物（GTP γ S）的能力。方法使用纯化的重组G α（i 1）测定氟烷对GTP酶特异性活性和[35 S] GTP γ S结合的影响。在不同的实验中，[35 S] GTP γ S与粗气道平滑肌膜制备物中的G α（i）的结合在氟烷存在和不存在的情况下使用免疫沉淀技术进行测定。（平均值+/-SD）摩尔P（i）摩尔G α（i1）-1 min-1（对照条件下）和0.035+/-0.015摩尔P（i）摩尔G α（i1）-1 min-1（存在1.1+/-0.2 mm氟烷时），差异不显著。与[35 S] GTP γ S结合的重组Ga（i 1）的摩尔分数在10和20 min时分别为0.49+/-0.02和0.60+/-0.02。添加氟烷（1.26+/-0.07 mm）不会显著改变这些值。氟烷不影响[35 S] GTP γ S与气道平滑肌膜组分中Ga（i）亚单位的结合，如使用免疫沉淀法测量的。使用苏拉明，GTP binding. CONCLUSION的抑制剂的测定的有效性得到证实，这些结果表明，氟烷，抑制受体激活的G α（i）偶联通路在完整的气道平滑肌，必须功能性靶向的G蛋白偶联受体复合物的一个组成部分以外的G α（i）。
BACKGROUNDReceptor-mediated increases in the force produced by airway smooth muscle are attenuated by anesthetics such as halothane. Guanosine 5'-triphosphate (GTP) binding protein alpha subunits (Galpha (i)) are known to participate in the regulation of force in airway smooth muscle. The authors hypothesized that halothane would inhibit the ability of Galpha (i) subunits to bind a nonhydrolyzable analog of GTP (GTPgammaS).METHODSThe effect of halothane on both GTPase-specific activity and [35S] GTPgammaS binding were assayed using purified, recombinant Galpha (i1). In separate experiments,[35S] GTPgammaS binding to Galpha (i) in crude airway smooth muscle membrane preparations was assayed using an immunoprecipitation technique in the presence and absence of halothane.RESULTSThe steady state GTPase-specific activity of the recombinant Galpha (i1) was 0.033+/-0.018 (mean+/-SD) mole P (i) mole Galpha (i1)-1 min-1 under control conditions and 0.035+/-0.015 mole P (i) mole Galpha (i1)-1 min-1 in the presence of 1.1+/-0.2 mm halothane, a difference that is not significant. The mole fractions of recombinant Galpha (i1) bound to [35S] GTPgammaS were 0.49+/-0.02 and 0.60+/-0.02 at 10 and 20 min, respectively. The addition of halothane (1.26+/-0.07 mm) did not significantly change these values. Halothane did not affect the binding of [35S] GTPgammaS to Galpha (i) subunits in membrane fractions of airway smooth muscle as measured using immunoprecipitation. Validity of the assays was confirmed using suramin, an inhibitor of GTP binding.CONCLUSIONThese results suggest that halothane, which inhibits receptor-activated Galpha (i)-coupled pathways in intact airway smooth muscle, must functionally target a component of the G protein-coupled receptor complex other than Galpha (i).