Plasma membrane ATPase of yeast. Comparative inhibition studies of the purified and membrane-bound enzymes.

The purified and lipid-reconstituted plasma m e m b r a n e A T P a s e o f the y e a s t Schizosaccharomyces pombe, which is believed to function as an electrogenic proton pump, is shown to be sensitive to seven different inhibitors. Most of the inhibition kinetics yields straight lines when analyzed by the graphical methods of Lineweaver-Burk ( l / u uersus l / [q) , Dixon ( l /u uersus [Q), and W e b b ( l / i u e m u l/[Q), indicating classic Michaelis-Menten mechanisms and noninteracting binding sites for the substrate MgATP. A new ATPase inhibitor, octylaminolester (ester derivative of erythro-p-(isopropy1thio)-a-[1-(octy1amino)-ethyl]benzylalcohol), yields strict competitive inhibition pattern. Miconazole nitrate (1(2-(2,4-dichlorophenyl)-2 [(2,4-dichlorophenyl) methoxy ethyl} -lH-imidazole nitrate) is also a strict competitive inhibitor. Purified Dio-9, the most powerful inhibitor among those tested, is classified as a presumably competitive inhibitor. Or thovanadate and N,Wdicyclohexylcarbodiimide exhibit strict noncompetit ive inhibition pattern. p-Hydroxymercuribenzoate irreversibly inhibits the ATPase act ivi ty and NaF exhibits a complex inhibition pattern, depending on the [M&+]/[MgATP] ratio. A general similarity is observed in the responses of the membrane-bound and purified ATPases to all inhibitors tested. It is concluded that the integrity of the ATPase activity is not appreciably modified by its solubilization by lysolecithin and the subsequent purification and lipid reconstitution procedures. A t least four of these ATPase inhibitors: N,N"dicyclohexylcarbodiimide, pure Dio-9, miconazole, and octylaminolester were tested in uiuo. They all induce a rapid efflux of K' accompanied by the stoichiometric influx of H' when added at pH 4.5 to intact cells incubated in the presence of glucose. It is concluded that the plasma membrane ATPase activity tightly controls not only the cellular fluxes of H' but also of K'.