Kaestner K H, Randall S K, Sze H
Department of Botany, University of Maryland, College Park 20742.
J Biol Chem. 1988 Jan 25;263(3):1282-7.
The inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was used to probe the structure and function of the vacuolar H+-translocating ATPase from oat roots (Avena sativa var. Lang). The second-order rate constant for DCCD inhibition was inversely related to the concentration of membrane, indicating that DCCD reached the inhibitory site by concentrating in the hydrophobic environment. [14C]DCCD preferentially labeled a 16-kDa polypeptide of tonoplast vesicles, and the amount of [14C]DCCD bound to the 16-kDa peptide was directly proportional to inhibition of ATPase activity. A 16-kDa polypeptide had previously been shown to be part of the purified tonoplast ATPase. As predicted from the observed noncooperative inhibition, binding studies showed that 1 mol of DCCD was bound per mol of ATPase when the enzyme was completely inactivated. The DCCD-binding 16-kDa polypeptide was purified 12-fold by chloroform/methanol extraction. This protein was thus classified as a proteolipid, and its identity as part of the ATPase was confirmed by positive reaction with the antibody to the purified ATPase on immunoblots. From the purification studies, we estimated that the 16-kDa subunit was present in multiple (4-8) copies/holoenzyme. The purification of the proteolipid is a first step towards testing its proposed role in H+ translocation.
抑制剂N，N'-二环己基碳二亚胺（DCCD）被用于探究燕麦根（燕麦品种Lang）液泡H⁺转运ATP酶的结构与功能。DCCD抑制的二级速率常数与膜浓度呈反比，这表明DCCD通过在疏水环境中富集到达抑制位点。[¹⁴C]DCCD优先标记液泡膜囊泡的一种16 kDa多肽，并且与该16 kDa肽结合的[¹⁴C]DCCD量与ATP酶活性的抑制呈正比。先前已表明一种16 kDa多肽是纯化的液泡膜ATP酶的一部分。正如从观察到的非协同抑制所预测的那样，结合研究表明当酶完全失活时，每摩尔ATP酶结合1摩尔DCCD。通过氯仿/甲醇提取将结合DCCD的16 kDa多肽纯化了12倍。因此该蛋白质被归类为一种蛋白脂质，并且通过免疫印迹上与纯化的ATP酶抗体的阳性反应证实了其作为ATP酶一部分的身份。从纯化研究中，我们估计16 kDa亚基以多个（4 - 8）拷贝/全酶的形式存在。蛋白脂质的纯化是测试其在H⁺转运中所提出作用的第一步。
