Large-scale isolation of the Neurospora plasma membrane H+-ATPase.
Large-scale isolation of the Neurospora plasma membrane H+-ATPase.
复制标题
脉孢菌质膜 H-ATP 酶的大规模分离。
DOI：
10.1016/0003-2697(84)90784-x
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发表时间：
1984
影响因子：
2.9
通讯作者：
Scarborough,GA
中科院分区：
文献类型：
--
作者：
Smith,R;Scarborough,GA
描述了一种纯化相对大量粗神经孢子虫质膜质子易位atp酶的方法。在o2条件下培养的神经孢子菌无壁sl株细胞以增加细胞产量，用豆豆蛋白A稳定质膜，在脱氧胆酸中匀浆，得到的裂解液在13500 g下离心。获得的颗粒几乎完全由豆豆蛋白a稳定的质膜片组成，富含H+- atp酶。α-甲基甘露糖苷处理后去除大部分刀豆蛋白A，再用溶卵磷脂处理膜，溶卵磷脂优先提取H+- atp酶。通过甘油密度梯度沉淀法纯化溶卵磷脂溶解的atp酶，得到约50mg酶，根据考马斯蓝染色凝胶的定量密度测定，该酶不含91%的其他蛋白质。该阶段酶在30℃下的比活性约为33 μmol pirelease /min/mg蛋白质。第二个甘油密度梯度沉降步骤产生的atp酶纯度约为97%，比活性约为35。对于不需要催化活性atp酶的化学研究或其他研究，可以通过在Sephacryl S-300上对十二烷基硫酸钠分解、梯度纯化的atp酶进行排斥层析制备几乎纯的酶。
A method for the purification of relatively large quantities of the Neurospora crassa plasma membrane proton translocating ATPase is described. Cells of the cell wall-less sl strain of Neurospora grown under O2to increase cell yields are treated with concanavalin A to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. The pellet obtained consists almost solely of concanavalin A-stabilized plasma membrane sheets greatly enriched in the H+-ATPase. After removal of the bulk of the concanavalin A by treatment of the sheets with α-methylmannoside, the membranes are treated with lysolecithin, which preferentially extracts the H+-ATPase. Purification of the lysolecithin-solubilized ATPase by glycerol density gradient sedimentation yields approximately 50 mg of enzyme that is 91% free of other proteins as judged by quantitative densitometry of Coomassie blue-stained gels. The specific activity of the enzyme at this stage is about 33 μmol of Pireleased/min/mg of protein at 30°C. A second glycerol density gradient sedimentation step yields ATPase that is about 97% pure with a specific activity of about 35. For chemical studies or other investigations that do not require catalytically active ATPase, virtually pure enzyme can be prepared by exclusion chromatography of the sodium dodecyl sulfate-disaggregated, gradient-purified ATPase on Sephacryl S-300.