Structure of yeast plasma membrane H+-ATPase: Comparison of activated and basal-level enzyme forms

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Abstract

Plasma membrane H+ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose- metabolising cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Structure of the two enzyme forms and the effects of β,γ/- imidoadenosine 5'-triphosphate (AMP-PNP) and of diethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. IR spectra revealed the presence of two populations of α-helices with different exposure to the solvent in both the A-ATPase and the B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two α-helix populations. Thermal denaturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while DES destabilised both enzyme forms. IR spectra showed that 60% of the amide hydrogens were exchanged for deuterium in both forms at 20°C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55°C for both enzyme forms, while in the presence of DES it was observed at lower temperatures. The data do not contradict the possibility that the activation of H-+-ATPase is due to the C-terminus of the enzyme dissociating from the ATP-binding site which is covered by it in the less active form.

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Tanfani, F., Lapathitis, G., Bertoli, E., & Kotyk, A. (1998). Structure of yeast plasma membrane H+-ATPase: Comparison of activated and basal-level enzyme forms. Biochimica et Biophysica Acta - Biomembranes, 1369(1), 109–118. https://doi.org/10.1016/S0005-2736(97)00216-2

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