牛脑V型质子转运ATP酶复合体依赖于温度的活性变化

Temperature-Dependent Changes of the Activity in the ATPase Complex of Clathrin Coated Vesicles from Bovine Brain

在ＫＣｌ介质中牛脑Ｖ－型质子转运ＡＴＰ酶复合体活力温度的Ａｒｒｈｅｎｉｕｓ图在３３℃附近呈现明显的折点，同样做其Ｎ－［１－芘］马来酰亚胺（Ｎ－［１－Ｐ］Ｍ）的荧光－温度的Ａｒｒｈｅｎｉｕｓ图，发现其折点温度也为３３℃，当加入１００μｍｏｌ／ＬＮＥＭ（Ｎ－ｅｔｈｙｌｍａｌｅｉｍｉｄｅ），ＡＴＰ酶复合体活力部分被抑制后的Ａｒｒｈｅｎｉｕｓ图折点下降为２７℃，加入０．７５－０．８５ｍｏｌ／Ｌ尿素则活力的Ａｒｒｈｅｎｉｕｓ图的折点变为３０℃。加入６％（Ｖ／Ｖ）的乙醇后，活力的Ａｒｒｈｅｎｉｕｓ图的折点上升为３８℃。加入ＮＥＭ，尿素和乙醇的内源荧光和Ｎ－［１－Ｐ］Ｍ标记的荧光测定，表明它们确实引起了牛脑Ｖ－型质子转运复合体构象的改变，这表明引起构象变化配基的加入，可改变牛脑Ｖ－型质子转运ＡＴＰ酶复合体的Ａｒｒｈｅｎｉｕｓ图折点温度，也说明牛脑Ｖ－型质子转运ＡＴＰ酶复合体Ａｒｒｈｅｎｉｕｓ图折点与酶复合体的构象直接相关。

A discontinuity at 33℃ is observed in the Arrhenius plot for ATPase hydrolysis by the H+-translocating ATPase complex of clathrin-coated vesicles in the reaction media of KCl. A break at 33℃ is also appeared in the Arrhenius plot for flourescence of N-(1-pyrenly)-malcimide-labelled ATPase complex, which coincides with that where a discontinuity in the Arrhenius plot of ATPase activity is found. When the ATPase complex treated with 100 μmol/L NEM, 0.75-0. 85mol/L Urea, or 6%(V/V) Ethyl alcohol,the break in the Arrhenius plot of the ATPase complex results in shifting, and a discontinuity at 27℃,30℃ and 38℃ is observed respectively. The measurements of the intrinsic tryptophan fluorescence and the flourescence labelled with N- (1-pyrenly)-maleimide of the ATPase complex treated with NEM, Urea, or Ethyl alcohol show changes of conformation of the ATPase complex.These results indicate that the addition of ligands leading to change of conformation can shift the break in the Arrhenius plot of the ATPase complex of clathrincoated vesicles, and that the discontinuity in Arrhenius plot of the ATPase complex is correlated with conformation of the ATPase complex directly.