Smith R, Scarborough G A
Anal Biochem. 1984 Apr;138(1):156-63. doi: 10.1016/0003-2697(84)90784-x.
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 O2 to 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 alpha-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 mumol of P1 released/min/mg of protein at 30 degrees 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.
本文描述了一种纯化相对大量粗糙脉孢菌质膜质子转运ATP酶的方法。在氧气条件下培养无细胞壁的粗糙脉孢菌sl菌株以提高细胞产量，用伴刀豆球蛋白A处理细胞以稳定质膜，然后在脱氧胆酸盐中匀浆，所得裂解物在13,500g下离心。得到的沉淀几乎完全由富含H⁺-ATP酶的伴刀豆球蛋白A稳定的质膜片组成。用α-甲基甘露糖苷处理这些膜片以去除大部分伴刀豆球蛋白A后，再用溶血卵磷脂处理，溶血卵磷脂优先提取H⁺-ATP酶。通过甘油密度梯度沉降法纯化溶血卵磷脂溶解的ATP酶，可得到约50mg的酶，根据考马斯亮蓝染色凝胶的定量光密度测定，该酶不含其他蛋白质的比例为91%。在30℃时，该酶在此阶段的比活性约为每分钟每毫克蛋白质释放33μmol的P1。第二步甘油密度梯度沉降可得到纯度约为97%、比活性约为35的ATP酶。对于不需要催化活性ATP酶的化学研究或其他调查，可以通过在Sephacryl S-300上对十二烷基硫酸钠解离的、梯度纯化的ATP酶进行排阻色谱来制备几乎纯的酶。
