Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity.
Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity.
复制标题
通过寡聚状态控制细胞膜外源 ATP 酶：分子间交联调节 ATP 酶活性。
DOI：
10.1021/bi960563g
复制
发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
Kirley,TL
中科院分区：
文献类型：
--
作者：
Stout,JG;Kirley,TL
胞外atp酶（外atp酶）是一种具有异常高特异性活性的二价阳离子依赖性核苷三磷酸酶。单克隆抗体，先前描述的[Stout， J. G., Strobel， R. S., & Kirley， T. L. (1995)J. biol。Chem.270, 11845−11850]和新生成的多克隆抗体，都是针对鸡胗外泌atpase培养的，它们与哺乳动物外泌atpase交叉反应的能力被评估，并被用作特异性免疫化学探针来识别非交联和交联的外泌atpase。与兔骨骼肌外链atp酶不同的是，鸡肫平滑肌外链atp酶与3,3′-二硫比斯（磺基琥珀酰酰丙酸）（DTSSP）和二硫比斯（琥珀酰酰丙酸）（DSP）交联后，酶活性增加，相当于约130 kDa的免疫反应带（被认为是外链atp酶的同型二聚体）增加，同时约66 kDa的免疫反应带（外链atp酶的单分子）减少。体外三磷酸腺苷酶在鸡、大鼠、小鼠、家兔和猪中进行了免疫化学鉴定。有趣的是，在非还原条件下，大鼠和猪（不像鸡和兔）的外链atp酶活性在Western blots上表现为一个约200 kDa的免疫反应带，被认为是分子间二硫连接的外链atp酶同源三聚体。用三种不同的单克隆抗体对不同的大鼠组织进行非还原性Western blot分析，这些单克隆抗体识别66 kDa的鸡胗外泌atp酶单体，从而加强了这条200 kDa的条带确实代表三聚体外泌atp酶的假设。还原后，发现所有物种的外链atp酶单体约为66 kDa。外三磷酸腺苷酶四元结构稳定性的差异可能解释了所观察到的外三磷酸腺苷酶酶学性质的物种差异。分子间二硫键似乎是一种物种特异性的方式来稳定原生的，活跃的外三聚体- atp酶的四元结构（同质三聚体）。基于获得的数据，以及本实验室和其他实验室先前的数据，提出了一种假设来解释各种试剂（包括洗涤剂、化学交联剂、凝集素、抗体和小分子抑制剂）对外三磷酸腺苷酶活性的调节。有人提出，稳定外三磷酸腺苷酶低聚物的药物和条件会刺激酶活性，而破坏外三磷酸腺苷酶同聚物的药物和条件会抑制外三磷酸腺苷酶。
The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995)J.Biol. Chem.270, 11845−11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3‘-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a ≈130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a ≈66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at ≈200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be ≈66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.