In vivo activation of yeast plasma membrane H+-ATPase by ethanol: effect on the kinetic parameters and involvement of the carboxyl-terminus regulatory domain.
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 Mg2+); the rectangular hyperbolic function was solved using iterative procedures. Ethanol-induced stimulation of plasma membrane H+-ATPase activity was associated to the increase of Vmax whereas the Km 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.
Monteiro
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GA
Laboratório de Engenharia Bioquímica, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1096 Lisboa Codex, Portugal.
Sá-Correia
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I
D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D017077:Culture Media, Conditioned; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D005947:Glucose / Q000494:pharmacology; D007700:Kinetics; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013012:Sorbitol / Q000494:pharmacology
D017077:Culture Media, Conditioned; D000431:Ethanol; D013012:Sorbitol; D006180:Proton-Translocating ATPases; D005947:Glucose