Large-scale isolation of the Neurospora plasma membrane H+-ATPase.
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.
D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A; D008854:Microscopy, Electron; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*
D003208:Concanavalin A; D006180:Proton-Translocating ATPases