Xie X S, Crider B P, Stone D K
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121.
J Biol Chem. 1993 Nov 25;268(33):25063-7.
An activator of the clathrin-coated vesicle proton translocating ATPase has been purified 1600-fold from bovine brain. The activator, which requires detergent (polyoxyethylene 9-lauryl ether) for release from clathrin-coated vesicles, is heat-stable, trypsin-sensitive, and has an apparent molecular mass of about 6 kDa as determined by high performance liquid chromatography. The activator stimulates the purified H(+)-ATPase of coated vesicles over 50-fold under acidic conditions. Similarly, the activator stimulates proton pumping catalyzed by the reconstituted proton pump. Importantly, this stimulation of proton pumping is observed only when the activator is reconstituted into the interior of the proteoliposomes. Moreover, the activator protein is demonstrated to protect, and co-sediment with, purified proton pump during glycerol gradient centrifugation performed in the presence of ATP. These observations support the notion that this activator serves to determine the pH set point of acidic endomembranes through interactions with the transmembranous sectors of the proton pump.
一种网格蛋白包被囊泡质子转运ATP酶的激活剂已从牛脑中纯化出来，纯化倍数达1600倍。该激活剂需要去污剂（聚氧乙烯9-月桂基醚）才能从网格蛋白包被囊泡中释放出来，它对热稳定，对胰蛋白酶敏感，通过高效液相色谱测定其表观分子量约为6 kDa。在酸性条件下，该激活剂可将纯化的包被囊泡H(+)-ATP酶的活性刺激50倍以上。同样，该激活剂也能刺激重组质子泵催化的质子泵出。重要的是，只有当激活剂重组到蛋白脂质体内部时，才会观察到这种对质子泵出的刺激。此外，在ATP存在下进行甘油梯度离心时，激活剂蛋白被证明能保护纯化的质子泵并与之共沉降。这些观察结果支持了这样一种观点，即这种激活剂通过与质子泵的跨膜区段相互作用来确定酸性内膜的pH设定点。
