Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms.
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 beta, gamma-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 alpha-helices with different exposure to the solvent in both the A-ATPase and B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-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 degrees C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55 degrees 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.
Tanfani
F
F
Institute of Biochemistry, Medical School, University of Ancona, Italy.
Lapathitis
G
G
Bertoli
E
E
Kotyk
A
A
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D004054:Diethylstilbestrol / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared
D004791:Enzyme Inhibitors; D000266:Adenylyl Imidodiphosphate; D004054:Diethylstilbestrol; D006180:Proton-Translocating ATPases