STRUCTURE OF YEAST PLASMA-MEMBRANE H-ATPASE - COMPARISON OF ACTIVATEDAND BASAL-LEVEL ENZYME FORMS()

Plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae was is olated and purified in its two forms, the activated A-ATPase from gluc ose-metabolising cells, and the basal-level B-ATPase from cells with e ndogenous metabolism only. Structure of the two enzyme forms and the e ffects of beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP) and of d iethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. I R spectra revealed the presence of two populations of alpha-helices wi th different exposure to the solvent in both the A-ATPase and the B-AT Pase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-helix populations. Thermal de naturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while D ES destabilised both enzyme forms. IR spectra showed that 60% of the a mide hydrogens were exchanged for deuterium in both forms at 20 degree s C. The remaining 40% were exchanged at higher temperatures. The maxi mum amount of H/D exchange was observed at 50-55 degrees C for both en zyme forms, while in the presence of DES it was observed at lower temp eratures. The data do not contradict the possibility that the activati on of H+-ATPase is due to the C-terminus of the enzyme dissociating fr om the ATP-binding site which is covered by it in the less active form . (C) 1998 Elsevier Science B.V.