Stout J G, Kirley T L
Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575, USA.
Biochemistry. 1996 Jun 25;35(25):8289-98. doi: 10.1021/bi960563g.
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 approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 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 approximately 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 approximately 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.
细胞外ATP酶（ecto-ATPase）是一种依赖二价阳离子的核苷三磷酸酶，具有异常高的比活性。先前已描述过的单克隆抗体[Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845 - 11850]以及新产生的多克隆抗体，均是针对鸡砂囊ecto-ATPase制备的，评估了它们与哺乳动物ecto-ATP酶交叉反应的能力，并用作特异性免疫化学探针来鉴定未交联和交联的ecto-ATP酶。与先前用兔骨骼肌ecto-ATP酶获得的结果不同，用3,3'-二硫代双（磺基琥珀酰亚胺丙酸酯）（DTSSP）和二硫代双（琥珀酰亚胺丙酸酯）（DSP）交联鸡砂囊平滑肌ecto-ATP酶会增加该酶的活性，这与一条约130 kDa免疫反应带的增加相对应，该条带被认为是ecto-ATP酶同型二聚体，同时一条约66 kDa免疫反应带（ecto-ATP酶单体）的强度随之降低。在鸡、大鼠、小鼠、兔和猪中均通过免疫化学方法鉴定出了ecto-ATP酶。有趣的是，在非还原条件下，大鼠和猪（与鸡和兔不同）的ecto-ATP酶活性在蛋白质印迹上表现为一条约200 kDa的免疫反应带，该条带被认为是分子间二硫键连接的ecto-ATP酶同型三聚体。用三种识别66 kDa鸡砂囊ecto-ATP酶单体的不同单克隆抗体对各种大鼠组织进行非还原蛋白质印迹分析，强化了这一假设，即这条200 kDa的条带确实代表三聚体ecto-ATP酶。还原后，在所有检测的物种中均发现ecto-ATP酶单体约为66 kDa。ecto-ATP酶四级结构稳定性的差异可能解释了所观察到的ecto-ATP酶酶学性质的物种差异。分子间二硫键似乎是稳定天然活性ecto-ATP酶四级结构（同型三聚体）的物种特异性方式之一。基于所获得的数据以及本实验室和其他实验室先前的数据，提出了一个假设来解释包括去污剂、化学交联剂、凝集素、抗体和小分子抑制剂在内的多种试剂对ecto-ATP酶活性的调节作用。有人提出，稳定ecto-ATP酶寡聚体的试剂和条件会刺激酶活性，而使ecto-ATP酶同型寡聚体不稳定的试剂和条件则会抑制ecto-ATP酶。
