Ravindra R, Grosvenor C E
Department of Medicine, East Carolina University, Greenville, North Carolina 27858.
Endocrinology. 1992 Jan;130(1):263-8. doi: 10.1210/endo.130.1.1727702.
A GTPase assay was employed to determine the relative proportions of the enzymatic activity in soluble and polymerized tubulin pools in the anterior pituitary lobe of the lactating rat. The GTPase activity in the tubulin fractions was estimated in 25-50 micrograms protein using [gamma-32P]GTP. The liberation of inorganic phosphate (Pi) was proportional to the protein concentration with either of the tubulin fractions. The enzymatic activity appeared to reach equilibrium by 1 min. Antitubulin antibodies inhibited the enzymatic activity in a concentration-dependent manner in both the tubulin fractions; at a final dilution of 1:2000 the antibody maximally inhibited the enzyme activity in both the tubulin fractions by 39-44%. After establishing the optimal conditions for the GTPase assay, the effect of suckling on pituitary GTPase activity was studied. Soluble and polymerized tubulin fractions were prepared from anterior pituitaries obtained from lactating rats killed after suckling for 30, 60, and 90 min; GTPase activity was assayed in both the tubulin fractions in the absence of antitubulin antibody. Compared to the nonsuckled control, suckling for 60 and 90 min stimulated the enzymatic activity in the soluble tubulin fraction by 80% and 44%, respectively (P less than 0.05). The enzymatic activity in the polymerized tubulin fraction increased by 30% at 60 min and decreased by about 20% at 90 min (P less than 0.05). The suckling-stimulated GTPase activity in the two pituitary fractions cannot be attributed to tubulin alone since there are other proteins also capable of hydrolyzing GTP. Therefore, GTPase activity was assayed in the pituitary tubulin fractions in the presence of antitubulin antibody (1:2000 dilution); tubulin-GTPase activity is the difference between the activity assayed in the absence of the antibody and that which was determined in the presence of the antibody. In the soluble tubulin fraction, tubulin-GTPase activity increased by 166% at 30 min suckling (P less than 0.05), decreased by 40% at 60 min (P less than 0.05), and again increased by 148% at 90 min (P less than 0.05). In the polymerized tubulin fraction, the enzyme activity decreased by 82% at 30 min (P less than 0.05), increased by 742% at 60 min (P less than 0.05), and again decreased by 95% at 90 min (P less than 0.05). Thus, an inverse relationship between tubulin-GTPase activities in the two pituitary fractions was observed and provides further evidence in support of our hypothesis that microtubules are recruited to transport PRL granules from the Golgi apparatus to the plasma membrane.
采用GTP酶分析来确定泌乳大鼠垂体前叶中可溶性和聚合微管蛋白库中酶活性的相对比例。使用[γ-32P]GTP在25 - 50微克蛋白质中估计微管蛋白组分中的GTP酶活性。无机磷酸盐（Pi）的释放与任一微管蛋白组分中的蛋白质浓度成正比。酶活性在1分钟时似乎达到平衡。抗微管蛋白抗体在两种微管蛋白组分中均以浓度依赖性方式抑制酶活性；在终浓度为1:2000时，抗体在两种微管蛋白组分中最大程度地抑制酶活性39 - 44%。在确定GTP酶分析的最佳条件后，研究了哺乳对垂体GTP酶活性的影响。从哺乳30、60和90分钟后处死的泌乳大鼠的垂体前叶中制备可溶性和聚合微管蛋白组分；在不存在抗微管蛋白抗体的情况下，对两种微管蛋白组分进行GTP酶活性分析。与未哺乳的对照组相比，哺乳60分钟和90分钟分别使可溶性微管蛋白组分中的酶活性提高了80%和44%（P < 0.05）。聚合微管蛋白组分中的酶活性在60分钟时增加了30%，在90分钟时下降了约20%（P < 0.05）。两种垂体组分中哺乳刺激的GTP酶活性不能仅归因于微管蛋白，因为还有其他蛋白质也能够水解GTP。因此，在存在抗微管蛋白抗体（1:2000稀释）的情况下，对垂体微管蛋白组分进行GTP酶活性分析；微管蛋白 - GTP酶活性是在不存在抗体时测定的活性与在存在抗体时测定的活性之间的差值。在可溶性微管蛋白组分中，哺乳30分钟时微管蛋白 - GTP酶活性增加了166%（P < 0.05），60分钟时下降了40%（P < 0.05），90分钟时再次增加了148%（P < 0.05）。在聚合微管蛋白组分中，酶活性在30分钟时下降了82%（P < 0.05），60分钟时增加了742%（P < 0.05），90分钟时再次下降了95%（P < 0.05）。因此，观察到两种垂体组分中微管蛋白 - GTP酶活性之间呈负相关，这为我们的假设提供了进一步的证据，即微管被募集来将催乳素颗粒从高尔基体运输到质膜。
