43] Large-scale purification of plasma membrane H+-ATPase from a cell wall-less mutant of Neurospora crassa

Abstract

Publisher Summary This chapter discusses large-scale purification of plasma membrane H + -ATPase from a cell wall-less mutant of Neurospora crassa . The plasma membrane of the filamentous fungus, Neurospora crassa , contains an electrogenic, proton-translocating ATPase, which functions to generate a transmembrane electrochemical protonic potential difference that is utilized by substrate-specific porters, to energize the cellular accumulation or extrusion of a variety of specific ions, nutrients, and metabolites. Two original published methods for the purification of this ATPase involved isolation of plasma membranes, solubilization of the ATPase with detergents, and subsequent purification of the enzyme by Sepharose CL-6B chromatography, and/or glycerol density gradient sedimentation. Both of these methods yielded reasonably pure ATPase, but they were both fairly time-consuming and the yields were in the range of only a few hundred micrograms. The cell breakage problem, associated with the use of wild-type Neurospora, is obviated by the use of a cell wall-less mutant. Cells of the cell wall-less mutant are treated with concanavalin A (Con A), which coats and stabilizes the plasma membrane.