Beruflich Dokumente
Kultur Dokumente
CHEMISTRY
Vol. 235, No. 5, May 1960
Printed
in U.S.A.
The Aerobic
Assimilation
of Glucose
by Yeast
Cells*
W. FALES
FRANK
for publication,
Sci-
2, 1959)
1255
November
Aerobic Assimilation
1256
CARBON
DIOXIDE
EXTRACELLULAR
CARBOHYDRATE
= 80
E
\ 40
RESULTS
CARBOHYDRATE
6 70
L,
z 20
s
I6
I?
.-
2
REACTJON
TIME.
3
HOURS
FIG.
1. The aerobic metabolism of glucose (2.0 mg per ml) by a
1% suspension of yeast cells : O---O,
aerated by rapidly passing
air through the suspension; O-0,
aerated by Warburg shaker;
broken
line, anaerobic.
The limiting concentration
for glucose
oxidation of this yeast was 1.6 X 10-a M or 29 mg per 100 ml (see
text).
The values recorded for fatty acid and cellular carbohydrate are the averages obtained with six separate experiments.
In these experiments, the differences obtained between the shaker
and bubbler were significant at all time intervals.
The statistical
probability
of no difference for the 30-minute cellular carbohydrate determinations
was p< 0.02; for the 75-minute fatty acid
determinations,
p = 0.02; and for the other determinations,
p<
0.01. The values recorded for the carbon dioxide, oxygen, and
extracellular
carbohydrate were those obtained in single experiments with each point being the average of duplicate determinations.
glucose solution to a 500-ml Erlenmeyer flask. When the Erlenmeyer flask was used, the shaking speed was diminished to 90
strokes per minute since the oxygen tension was maintained
at
the level observed in the Warburg vessels at this shaking rate.
A 200-ml chromatography
tube with a fritted glass base was
used as the reaction vessel in the experiments in which air was
The air was brought to the
bubbled through the suspension.
temperature of the water bath and saturated with water before
it entered the reaction vessel. Air was passed through the suspension at a rate of about 350 ml per minute.
Yeast suspension
and glucose solution were added in the volumes of 80 and 20 ml,
respectively.
Measurements
of the fluid volume indicated that
there was no net evaporation or condensation during the course
Also, there was no significant trapping of
of the experiments.
the cells in the fritted glass base since a constant cell count was
observed.
For each analysis, the bubbler and shaker experiments were carried out simultaneously
with aliquots of the same
yeast suspension and glucose solution.
The oxygen consumption
rate in the bubbler experiments was determined polarographically
by the method of Baumberger
(8). Aliquots of the suspension
were transferred to the polarographic
vessel for the measurements.
The total cellular and extracellular
carbohydrate
were determined by the anthrone method as previously described (5). The
supernatant fluid was immediately
separated from the cells by
centrifugation.
This immediate separation was found necessary
because there was a gradual diffusion of trehalose from the cells
after the reaction was stopped with HgC12. The total fatty acid
content of the cells was determined by the method of Klein (6).
Palmitic and oleic acids were utilized as the standards for the
calorimetric assay method of Kibrick and Skupp (9).
160
May
F. W. Fazes
1960
the inhibition of the synthesis by the uncoupling of phosphorylation is not readily understandable
since hexose phosphates are
intermediates
for both the synthesis and the fermentation
and
since the fermentation
was not inhibited.
It is of interest that
in the present study, there was a greater synthesis of both trehalose and glycogen in the bubbler than in the shaker experiments, and that the increased synthesis of the former was much
more marked than that of the latter.
Several hypotheses may be advanced for explaining the greater
fat synthesis that was observed in the shaker. More of the required reduced coenzyme may have been available for the fatty
acid synthesis at the lower oxygen tension (15). Also, the data
suggest that a portion of the alcohol, which accumulated in the
shaker, acted as a precursor for fat synthesis.
It is also possible
that the higher carbon dioxide tension in the shaker stimulated
fat synthesis (16, 17).
SUMMARY
3. GOTTSCHALK,
A., Australian
J. Exptl.
Biol. Med. Sci., 19, 211
(1941).
4. STIER,
T. J. B., Cold Spring
Harbor
symposia
on quantitative
biology,
Vol. 7, Long
Island
Biological
Association,
Cold
Spring
Harbor,
Long Island,
New York,
1939, p. 385.
5. FALES,
F. W., J. Biol. Chem., 193, 113 (1951).
6. KLEIN,
H. P., J. Bacterial.,
69, 620 (1955).
7. FALES,
F. W., AND BAUMBERGER,
J. P., J. Biol.
Chem., 173,
1 (1948).
8. BAUMBERGER,
J. P., Cold Spring
Harbor
symposia
on quantitative biology,
Vol. 7, Long
Island
Biological
Association,
Cold Spring
Harbor,
Long Island,
New York,
1939, p. 195.
9. KIBRICK,
A. C., AND SKUPP,
S. J., Arch. Biochem.
Biophys.,
44, 135 (1953).
10. LELOIR,
L. F., AND CARBIB,
E., J. Am. Chem. Sot., 76, 5445
(1953).
11. LELOIR,
L. F., AND CARDINI,
C. E., J. Am. Chem. SOL, 79,634O
(1957).
12. T~EVE~YAN,
W. E., MANN,
P. F. E., AND HARRISON,
J. S.,
Arch. Biochem.
BioDhus..
60. 81 (1954).
13. FALES,
F. W., J. BioZ. chek.,
aO2, i57 (i953).
14. SPIEGELMAN,
S., KAMEN,
M. D., AND SUSSMAN,
M., Arch.
Biochem.,
18, 409 (1948).
15. CHANCE,
B., J. Biol. Chem., 217, 383 (1955).
16. BRADY,
R. O., Proc. Natl.
Acad. Xci. U. S., 44, 993 (1958).
17. GIBSON,
D. M., TITCHENER,
E. B., AND WAKIL,
S. J., Biochim.
Biophys.
Acta, 30, 377 (1958).
The large differences in the metabolic pattern that were observed with the two modes of aeration were apparently induced
by differences in the gas tensions in the two systems since it would
appear that these were the only variables in the experimental
conditions.
The increased cellular carbohydrate
synthesis together with the decreased total oxygen consumption that was observed in the bubbler experiments, suggests a higher over-all
efficiency of oxidative phosphorylation
at the higher oxygen tension. This increased efficiency may not be due to a direct action
of oxygen on the coupling of phosphorylation
with oxidation.
In
the shaker experiments, the data suggest that the oxidation of
alcohol accounted for about half of the total oxygen consumption,
but in the bubbler experiments, oxidative fermentation
was inhibited.
The over-all efficiency of oxidative
phosphorylation
may be decreased when alcohol is an intermediate because of the
energy required for bringing the 2-carbon compound into the
oxidative pathway.
The energy requirements for cellular carbohydrate synthesis may be much higher than previously supposed.
Leloir and Carbib (10) have presented evidence that UDP-glucase and hexose phosphate are precursors for trehalose synthesis
in yeast. Also, UDP-glucose
and hexose phosphate may be
precursors for glycogen synthesis in yeast since there is evidence
that this may be the case in mammalian tissue (11) and since
Trevelyan et al. (12) have found that during fermentation
the
orthophosphate
and glucose l-phosphate
levels are maintained
in the cells at an unfavorable ratio for glycogen synthesis by the
Since there is a considerable glyyeast phosphorylase reaction.
cogen synthesis during fermentation,
the UDP-glucose pathway
for glycogen synthesis is suggested.
If UDP-glucose is indeed a
precursor for cellular carbohydrate
synthesis, energy is required
for the reformation of the UDP-glucose
as well as for the phosphorylation
of the sugar. This proposed requirement
of additional energy for cellular carbohydrate
synthesis is helpful in
explaining the effect of azide on alcoholic fermentation.
At an
appropriate azide concentration, the synthesis of cellular carbohydrate is completely inhibited without reducing the fermentation
rate (13). Spiegelman et al. (14) have presented evidence that
azide uncouples fermentative
phosphorylation.
A mechanism
for the azide inhibition is readily deduced with an energy-requiring step for the conversion of hexose phosphate to cellular carbohydrate.
However, if there were no energy-requiring
step, then
1257