Fischer-Schliebs E, Ball E, Berndt E, Besemfelder-Butz E, Binzel M L, Drobny M, Mühlenhoff D, Müller M L, Rakowski K, Ratajczak R
Technische Hochschule Darmstadt, Institut für Botanik, Germany.
Biol Chem. 1997 Oct;378(10):1131-9. doi: 10.1515/bchm.1997.378.10.1131.
Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.
对两种针对伽蓝菜（Kalanchoë daigremontiana）液泡膜H⁺-ATP酶全酶产生的抗血清（ATP88和ATP95），检测了它们与其他植物物种液泡膜H⁺-ATP酶亚基的交叉反应性。用针对冰叶日中花（Mesembryanthemum crystallinum）催化性液泡膜H⁺-ATP酶亚基A的抗血清，对来自长寿花（Kalanchoë blossfeldiana）、冰叶日中花、烟草（Nicotiana tabacum）、番茄（Lycopersicon esculentum）、柠檬（Citrus limon）、浮萍（Lemna gibba）、大麦（Hordeum vulgare）和玉米（Zea mays）的液泡膜H⁺-ATP酶进行免疫沉淀。如银染和用ATP88进行的蛋白质免疫印迹分析所示，亚基A、B、C、D和c存在于所有免疫沉淀的液泡膜H⁺-ATP酶中。相比之下，ATP95仅在长寿花、冰叶日中花、大麦和玉米中识别整套亚基。ATP95的这种不同交叉反应性表明某些液泡膜H⁺-ATP酶亚基存在结构差异。基于液泡膜富集囊泡对巴弗洛霉素A1敏感的ATP酶活性，以及可溶和免疫沉淀的液泡膜H⁺-ATP酶的量，测定了受试液泡膜H⁺-ATP酶的比ATP水解活性。这些结构差异与不同物种的液泡膜H⁺-ATP酶在一种给定的ATP水解测量测定条件下水解ATP的能力相关。有趣的是，与含有未被ATP95标记的亚基的液泡膜H⁺-ATP酶相比，显示亚基A、B、C、D和c与ATP95有交叉反应性的液泡膜H⁺-ATP酶表现出更高的比ATP水解速率。因此，在我们的测定条件下具有高周转率的液泡膜H⁺-ATP酶可能显示出将它们与低周转率的ATP酶区分开的共同结构特征。
