Energy
requirement for the anaerobic oxidation of acetate in baker´s
yeast
Stoppani, A.O.M., Bennun, A. and De Pahn, E.M.
Baker's yeast oxidizes acetate in the absence of oxygen or
in the presence of antimycin A, an inhibitor of electron transport.
The oxidation takes place mostly through the Krebs cycle and
is stimulated by glucose. The results summarized below establish
the necessity of high-energy phosphate for the anaerobic oxidation
of acetate.
The materials and methods employed were similar to those used
previously. For analysis of phosphate compounds, the cells
were extracted with 10% trichloro-acetic acid solution as
described before. Pi and the orthophosphate liberated after
acid hydrolysis of P7 were extracted with the isobutanol-benzene
mixture for chemical determination or counting of radioactivity.
P7 was hydrolyzed with 1 NH2SO4 for 7 min at 100°. In
order to determine ATP and ADP, nucleotides in the trichloroacetic
acid extract were adsorbed by stepwise addition of purified
Norit A (200 mg/5 ml of trichloroacetic acid extract). After
1 h at 30° the adsorbed nucleotides were centrifuged,
and eluted twice with ethanol-pyridine-water mixture (45:45:10,
v/v), first for 2 h at 37° and then for further 24 h.
The eluates were concentrated in vacuo and chromatographed
on Whatman No. 1 paper pre-treated with 10% Na2CO3; 1 N formic
acid; 0.01 M EDTA (pH 7), and ethanol (after each treatment
the respective reagent was washed out with distilled water).
The solvent system employed for chromatography was ethanol-ammonium
acetate. Radioactive [32P] nucleotides were located in the
chromatogram by radioautography and fluor¬escence under
a Mineralight lamp. After elution, the nucleotides were further
identified by cochromatography with pure specimens and the
concentration determined by absorbancy at 260 mµ.
Biochimica
et Biophysica Acta, 92, (1964), 176-178