In vivo activation of yeast plasma membrane H +-ATPase by ethanol: effect on the kinetic parameters and involvement of the carboxyl-terminus regulatory domain

Abstract

The in vivo activation of Saccharomyces cerevisiae plasma membrane H +-ATPase by ethanol was observed during ethanol-stressed cultivation or following the rapid incubation of cells with ethanol (6% (v/v)). Ethanol activated both the basal and the glucose-activated forms of the enzyme being the H +-ATPase fully activated by glucose (5% (w/v)) still additionally activable by ethanol. The kinetic parameters of ethanol-activated and non-activated H +-ATPase were calculated based directly on Michaëlis–Menten equation (with MgATP concentrations in the range 0.16–8.18 mM and 7.5 mM of free Mg 2+); the rectangular hyperbolic function was solved using iterative procedures. Ethanol-induced stimulation of plasma membrane H +-ATPase activity was associated to the increase of V max whereas the K m for MgATP increased. Results obtained with mutants constructed and used in previous studies envisaging the analysis of the molecular mechanisms underlying plasma membrane ATPase activation by glucose, external acidification and nitrogen starvation, suggested that the carboxyl-terminus (C-terminus) regulatory domain may also be involved in the in vivo activation by ethanol.