Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H+-ATPase in activated and basal-level enzyme preparations

Plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae was isolate d and purified in its two forms, the activated A-ATPase from glucose-metabo lizing cells, and the basal-level B-ATPase from cells with endogenous metab olism only. Using two-dimensional gel electrophoretic analysis, we showed t hat both enzyme preparations are actually mixtures of the non-active, i.e. non-phosphorylated, and the active, i.e. phosphorylated, forms of the enzym e. Previous deliberations suggesting that the B-ATPase displays some activi ty which is lower than that of A-ATPase were apparently wrong. It seems tha t, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stab le than the single forms. On the whole then, there apparently is a differen t ratio of the active and inactive forms and/or complexes between the two f orms present in all enzyme preparations. (C) 2001 Federation of European Bi ochemical Societies. Published by Elsevier Science B.V. All rights reserved .