Battastini A M, Oliveira E M, Moreira C M, Bonan C D, Sarkis J J, Dias R D
Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil.
Biochem Mol Biol Int. 1995 Oct;37(2):209-19.
The effect of different detergents on the ATPase and ADPase activities from synaptic plasma membrane were investigated. Triton X-100, deoxycholate, CHAPS, Nonidet, N-octylglucoside and C12E8, which is commonly used to solubilize plasma membrane proteins, easily inactivated the ATPase and ADPase activities, while digitonin was not harmful to the enzyme. Treatment of the synaptic plasma membrane from rat brain with 0.5% digitonin solubilizes 80% of the proteins and 50% and 60% of ATPase and ADPase, respectively, with the following characteristics: stimulation by Ca2+ in the millimolar range, insensitivity to ATPase inhibitors (ouabain, olygomicyn, orthovanadate), inhibition with sodium azide and NEM and broad substrate specificity for the hydrolysis of nucleoside di- and triphosphate. To further characterize the enzyme solubilized, polyclonal antibodies specific for ATP diphosphohydrolase from potato tuber were tested. Western blot showed that two electrophoretic bands with a molecular mass close to 60-70 kDa had cross-immunoreactivity with antibodies against potato apyrase. The results presented here demonstrate for the first time the solubilization of ATPase and ADPase activities with characteristics of a true ATP diphosphohydrolase from synaptic plasma membrane from rat brain and with cross-immunoreactivity with antibodies against potato apyrase.
研究了不同去污剂对突触质膜ATP酶和ADP酶活性的影响。常用于溶解质膜蛋白的Triton X-100、脱氧胆酸盐、CHAPS、Nonidet、N-辛基葡糖苷和C12E8容易使ATP酶和ADP酶活性失活，而洋地黄皂苷对该酶无害。用0.5%洋地黄皂苷处理大鼠脑突触质膜可溶解80%的蛋白质以及分别50%和60%的ATP酶和ADP酶，其具有以下特性：在毫摩尔范围内受Ca2+刺激，对ATP酶抑制剂（哇巴因、寡霉素、原钒酸盐）不敏感，受叠氮化钠和N-乙基马来酰亚胺抑制，对核苷二磷酸和三磷酸水解具有广泛的底物特异性。为进一步表征溶解的酶，测试了针对马铃薯块茎ATP二磷酸水解酶的多克隆抗体。蛋白质印迹显示，两条分子量接近60 - 70 kDa的电泳带与抗马铃薯焦磷酸酶抗体具有交叉免疫反应性。此处给出的结果首次证明了大鼠脑突触质膜中具有真正ATP二磷酸水解酶特性的ATP酶和ADP酶活性的溶解以及与抗马铃薯焦磷酸酶抗体的交叉免疫反应性。
