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Journal
of
General Microbiology
(1983),
129,
2125-2127.
Printed in Great Britain
2125
Glycolysis and Respiration
in
Yeasts: the Effect
of
Ammonium
Ions
Studied
by Mass Spectrometry
By DAVID LLOYD,*? BODIL KRISTENSEN
AND
HANS DEGN
Institute
of
Biochemistry, Odense University, Campusvej
55,
D
K-5230
Odense
M,
Denmark
(Received
2
February
1983;
revised
5
March
1983)
Addition of NH,+ in the presence of glucose to washed suspensions of Saccharomyces uvarum,
Schizosaccharomyces pombe or Candida utilis greatly increased glycolytic C02 production and
slightly stimulated respiration. In all three organisms the ammonium ion effect was
distinguishable from the effect of an uncoupler
of
aerobic energy conservation (carbonyl
cyanide m-chlorophenylhydrazone
;
CCCP). The Pasteur effect (aerobic inhibition of glycolysis)
in the fermentative yeasts also proceeded independently
of
the ammonium ion effect. Possible
control mechanisms are discussed.
INTRODUCTION
The Pasteur effect (inhibition by
O2
of the utilization of carbohydrates: Krebs, 1972) involves
the integration
of
several feedback mechanisms
;
especially important are the allosteric effectors
of phosphofructokinase. This enzyme is inhibited by ATP and citrate, and activated by AMP,
Pi,
NH,+
and
K+
(Ramaiah et al.,
1964;
Salas et al., 1965; Mavis
&
Stellwagen, 1970). In this
report we show that the addition of
NH,+
to washed non-proliferating cell suspensions
of
yeasts
stimulates glycolysis; this 'ammonium ion effect' is quite distinct from the stimulatory effect of
CCCP and from the Pasteur effect.
METHODS
Organisms and culture conditions. Candida utilis
NCYC 193 was maintained and grown on defined medium
containing
1
%
(w/v) glucose as described previously (Kader
&
Lloyd, 1979).
Saccharomyces uuarum
(formerly
S.
carlsbergensis)
NCYC 745 and
Schizosaccharomyces pombe
972 h- were maintained on 10% glucose/malt
extract/agar slopes and grown on defined medium containing
1
%
glucose as described by Mitchison (1970), but
with
0.5%
(NH4)2S04 as nitrogen source. All three yeasts were grown at
30
"C
with rotary shaking
(100
r.p.m.) as
200 ml cultures in
500
ml conical flasks. Organisms were counted in a Thoma haemocytometer slide (Hawkesly,
Lancing, Sussex,
U.K.)
after suitable dilution. They were harvested by centrifugation at 2000
g
for
2
min at
4
"C,
washed twice in
20
vol. of
15
mM-NaC1 and finally resuspended in
5
vol. of this solution.
Measurement
of
0,
and
C02
by mass spectrometry.
Concentrations
of
O2
and Cot were measured in yeast
suspensions (4.5
ml
in 10 mM-sodium citrate buffer, pH
4.0)
in a thennostated open reaction vessel of 7 ml total
volume using a quadrupole mass spectrometer type
QS
(VG Micromass, Winsford, Cheshire,
U.K.)
fitted with a
turbopump type TPN 100 (A. Pfeiffer Vacuum Tecknic, D-6334 Asslar,
F.R.G.).
Similar results were obtained
with other buffers, suggesting that the cells are not permeable to citrate. Details
of
the equipment have been
described previously (Lloyd
et al.,
1981). Mass per charge numbers 32 and 44 were scanned for
0,
and
CO,
respectively; half times for equilibration correspond to transfer constants
of
7.2
x
s-l
respectively and the scanning cycle was
0.3
s.
The gas phase was controlled by a calibrated digital gas mixer
(Lundsgaard
&
Degn, 1973). Steady-state rates
of
0,
consumption and
c02
evolution were calculated from steady-
state concentrations (Degn
et al.,
1980). The concentration
of
0,
in the buffer in equilibrium with atmospheric air
was assumed to be 240 p~. CO, measurement was calibrated by the injection of small amounts of 100 mM-
NaHC03 into the buffer in the reaction vessel.
s-l
and
5.8
x
7
Permanent address (for correspondence): Department
of
Microbiology, University College, Newport Road,
Cardiff
CF2
ITA,
U.K.
Abbreviation
:
CCCP, carbonyl cyanide m-chlorophenylhydrazone.
0022-1287/83/0001-1050 $02.00
0
1983 SGM
2126
D. LLOYD,
B.
KRISTENSEN AND
H.
DEGN
250
200
150
15C
h
3
0”
W
;
10
min
-
(4
Fig.
1.
O2
and C02 concentrations in washed cell suspensions
of
yeast: effect
of
ammonium ions.
(a)
Saccharomyces uvarum,
(b)
Schizosaccharornyces pornbe, (c) Candida utilis.
Cell concentrations in the
cultures at harvesting were
2
x
lo8,
8
x
lo7,
2
x
lo8
and in the reaction vessel
1-6
x
lo8,
8
x
lo7,
5
x
lo7
in
(a),
(b)
and
(c)
respectively;
11
mM-glUCOSe was present throughout. At arrows additions
were made or the composition
of
the
gas
stream entering the open system was altered as indicated.
----,
O2
(mass
32);
-,
COz
(mass
44).
RESULTS
AND
DISCUSSION
Figure 1 demonstrates the ammonium ion effect in the three yeasts. In the presence
of
11
mM-
glucose, switching the gas phase from
5%
O2
to
N2
stimulated
C02
evolution, and the effect
(Pasteur effect) was reversible. In
Saccharomyces
uvarurn
(Fig. 1
a)
addition of
0.5
mM-NH,Cl
(final concn)
to
the aerobic suspension gave a more pronounced stimulation
of
C02 evolution,
which reached a maximum after about
5
min. Respiration was also slightly stimulated
(<
5
%).
Ammonium
ion
efects
on
yeast fermentation
2127
Before C02 concentration had returned to its initial value the Pasteur effect was still
demonstrable
as
a stimulation of CO, production on making the suspension anaerobic. Addition
of NH,C1 anaerobically gave a burst of COz evolution (not shown). Stimulation
of
glycolysis
and respiration by an uncoupler of aerobic energy conservation (CCCP) was observed before or
after addition of NH4C1 (not shown). The ammonium ion effect was also observed in
Schizosaccharomycespombe
(Fig. 1
b),
and in
Candida
utilis
(Fig.
1
c),
although the Pasteur effect
was not evident in the latter. Control experiments indicated the specificity of stimulation by
NHZ; thus addition of
0.5
mM-NaC1 produced no response, whereas
0.5
~M-NH,OH in
potassium phosphate buffer gave similar effects to those observed for NH4Cl.
A possible explanation for the ammonium ion effect lies in the activation
of
phosphofructo-
kinase (Mavis
&
Stellwagen, 1970); consumption of ammonium ions to produce amino acids and
proteins may explain the transient nature of the effect. That the effects of anaerobiosis,
uncoupler and ammonium ions are additive suggests that maximal activation of phosphofructo-
kinase requires a combination of circumstances. Increased respiration in the presence of NH,+
may reflect increased energy demand for transport or biosynthetic reactions. These observations
may be related to those of Devine
&
Slaughter (1980) who reported that ethanol production was
accelerated in media containing high concentrations of ammonium ions.
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H.,
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S.,
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C.
&
ORMICKI,
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S.
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&
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C.
(1980). Effect
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