Solubilization and purification of the Neurospora plasma membrane H+-ATPase.
The electrogenic proton-translocating ATPase in the plasma membrane of Neurospora has been solubilized with lysolecithin and purified using a combination of gel filtration and density gradient centrifugation. Isolated plasma membrane vesicles are solubilized with lysolecithin in the presence of MgATP, vanadate, and chymostatin. The MgATP and vanadate are required to maintain the ATPase in an active form and the chymostatin prevents proteolytic degradation. Optimal solubilization of the H+-ATPase (approximately 70%) occurs at a lysolecithin/protein ratio of 3 (mg/mg). The lysolecithin extract is then passed through a Sepharose CL-6B column in the presence of lysolecithin, deoxycholate, MgATP, vanadate, dithiothreitol, and chymostatin. The bulk of the H+-ATPase activity elutes from this column as a relatively broad peak near the void volume. Sodium dodecyl sulfate-polyacrylamide gel analysis of the column fractions indicates that the H+-ATPase present in the first half of this peak is substantially separated from all other membrane proteins whereas the H+-ATPase in the second half of the peak is still significantly contaminated. Isopycnic glycerol density gradient centrifugation of the pooled fractions from each half of the column peak results in further purification of the H+-ATPase. Gradient fractions containing the H+-ATPase from the first half of the column peak are virtually free of any other membrane proteins and those from the second half of the column peak are only slightly contaminated. The overall yield of the H+-ATPase is at least 24% based upon enzyme activity, and the specific activity is approximately 10 mumol of Pi liberated/mg of protein/min.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D006180:Proton-Translocating ATPases; D012995:Solubility
D008244:Lysophosphatidylcholines; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases