Xie X S, Crider B P, Ma Y M, Stone D K
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121.
J Biol Chem. 1994 Oct 14;269(41):25809-15.
The vacuolar-type proton-translocating ATPase of clathrin-coated vesicles is composed of an integral membrane proton channel (VB) and a peripheral catalytic sector (VC). Native enzyme can catalyze the hydrolysis of both MgATP and CaATP and support proton pumping when reconstituted into liposomes. In contrast, isolated VC catalyzes only Ca(2+)-activated ATP hydrolysis and cannot support proton pumping when reconstituted into liposomes (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We now report that solubilized isolated VC can be reassembled with purified VB to restore properties of native enzyme, including Mg(2+)-activated ATP hydrolysis and proton-pumping capability. Investigation of this reassembly revealed that a heterodimer, composed of polypeptides of 50 and 57 kDa, stimulates Ca(2+)-activated ATPase activity of isolated VC 2-fold and Mg(2+)-activated ATPase activity catalyzed by the reassembled pump 9-fold. Moreover, this heterodimer stimulated proton transport by the reassembled pump > 20-fold. When separated from the proton pump, the dimer has no detectable kinase activity. Maximal stimulation occurs at a molar ratio of heterodimer to reassembled pump of 3, implying a structural, nonenzymatic mechanism. These data indicate that the 50-kDa and/or the 57-kDa polypeptide likely plays an essential and potentially regulatory role in the function of the proton-translocating ATPase of clathrin-coated vesicles.
网格蛋白包被小泡的液泡型质子转运ATP酶由一个整合膜质子通道（VB）和一个外周催化区（VC）组成。天然酶能催化MgATP和CaATP的水解，并且在重组到脂质体中时能支持质子泵浦。相比之下，分离得到的VC仅催化Ca(2+)激活的ATP水解，并且在重组到脂质体中时不能支持质子泵浦（谢，X.-S.，和斯通，D.K.（1988年）《生物化学杂志》263卷，9859 - 9867页）。我们现在报告，溶解的分离的VC可以与纯化的VB重新组装以恢复天然酶的特性，包括Mg(2+)激活的ATP水解和质子泵浦能力。对这种重新组装的研究表明，一个由50 kDa和57 kDa多肽组成的异二聚体，可使分离的VC的Ca(2+)激活的ATP酶活性提高2倍，使重组泵催化的Mg(2+)激活的ATP酶活性提高9倍。此外，这种异二聚体使重组泵的质子转运提高>20倍。当与质子泵分离时，该二聚体没有可检测到的激酶活性。在异二聚体与重组泵的摩尔比为3时出现最大刺激作用，这意味着一种结构上的、非酶促机制。这些数据表明，50 kDa和/或57 kDa多肽可能在网格蛋白包被小泡的质子转运ATP酶的功能中起重要且可能的调节作用。
