Purification of the Neurospora crassa plasma membrane H+-ATPase by high-pressure liquid chromatography in the presence of sodium dodecyl sulfate

Current methods for purifying the Mr 100,000 H+-ATPase from the plasma membrane of fungi and higher plants rely on detergent solubilization followed by density gradient centrifugation. The procedure yields catalytically active enzyme of high purity but takes several days, and the yields are low. For chemical studies on the primary structure of this enzyme, an alternative more rapid procedure was sought. In this paper a method which uses a high-performance gel filtration column in the presence of sodium dodecyl sulfate to purify nanomole quantities of the enzyme in only 30 min is described. With this procedure the enzyme was isolated free of other protein contaminants, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration was performed on a high-pressure liquid chromatograph equipped with a diode array spectrophotometric detector, allowing spectral analysis of the membrane proteins. An ultraviolet absorption spectrum of the plasma membrane Mr 104,000 H+-ATPase revealed an absorption peak at ca. 275 nm that is consistent with its content of aromatic amino acids.