Ravindra R, Aronstam R S
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta 30912.
Biochem Pharmacol. 1990 Aug 1;40(3):457-63. doi: 10.1016/0006-2952(90)90543-t.
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.
为了解微管蛋白（神经膜的一种组成成分）在信号转导过程中的作用，研究了抗微管蛋白抗体对与转导蛋白G蛋白相关的低Km GTP酶活性的影响，实验对象为大鼠纹状体。通过常规方法从纹状体制备细胞膜，并在含有0.2至2.0 microM未标记GTP的ATP再生缓冲液中，于37摄氏度下使用[γ-32P]GTP测定低Km GTP酶活性（EC 3.6.1.-）。GTP酶活性在长达30分钟内呈线性，且与蛋白质浓度成正比。多克隆抗微管蛋白抗体、抗α-微管蛋白抗体和抗β-微管蛋白抗体（10微克）可刺激G蛋白GTP酶活性。抗β-微管蛋白抗体（10微克）在每个时间点刺激GTP酶活性约60%，而10微克的抗α-微管蛋白或多克隆抗微管蛋白抗体在每个时间点仅刺激GTP酶活性20 - 30%。酶 - 底物相互作用的指标Vmax/Km比值，抗α-微管蛋白抗体使其仅增加26%，抗β-微管蛋白抗体使其增加52%；多克隆抗微管蛋白抗体不影响该比值。乙酰胆碱以阿托品敏感的方式刺激GTP酶活性。在100 microM时，乙酰胆碱刺激GTP酶活性约50%。多克隆抗微管蛋白、抗α-微管蛋白或抗β-微管蛋白抗体（10微克）增强了乙酰胆碱对GTP酶活性的刺激作用。抗微管蛋白抗体刺激低Km GTP酶活性并增强乙酰胆碱刺激作用的两种可能机制为：（1）通过抑制GTP与β-微管蛋白的结合，以及（2）通过消除微管蛋白对G蛋白或受体 - G蛋白相互作用的慢性抑制作用。
