Folia Microbiol. 27, 395--403 (1982)
Acidification Power: Indicator of Metabolic Activity
and Autolytic Changes in Saccharomyces cerevisiae
M . OPEKAROVA a n d K . SIGLER
Department of Cell Physiology, Institute of Microbiology.
Czechoslovak Academy of Sciences, 142 20 Prague 4
Received May 26, 1982
ABSTRACT. Acidification power, defined as the sum of the spontaneous p H change determined after
suspending yeast cells in water and the substrate-induced p H change after addition of glucose to the resulting suspension, reflects the level of cellular energy sources. Its use as an indicator of metabolic state of the
cells was tested during a 120-h aerobic starvation. Its changes coincided with changes in cell viability,
initial rate of endogenous oxygen consumption rate, cell ATP, extra- and intracellular buffering capacity,
and the ability of cell-free extract to produce acidity by glucose fermentation. I t was used as a sensitive
marker of metabolic changes occurring during starvation, on t r e a t m e n t with glycolytic and respiratory
inhibitors, and at elevated temperature.
A n y interference with the physiological state of the y e a s t cell (treatment with
various agents, change in cultivation conditions or nutritional regime, starvation)
brings a b o u t changes in metabolic processes. Our previous studies of proton extrusion
in y e a s t provided a useful common reference basis for these changes. Yeast cells
excrete protons either spontaneously on transfer into an unbuffered medium (Sigler
et al. 1980; K o t y k and Sigler 1981) or after addition of a suitable substrate (e.g. Sigler
et al. 1981a, b); the H + extrusion involves excretion of CO2 and organic acids K + / H +
exchange and the action of a plasma membrane H+-ATPase. Spontaneous H+-efflux
is sustained solely b y endogenous H + and energy sources while the substrate-induced
one is closely associated with the utilization of both endo- and exogenous sources.
Quantitative measurement of b o t h processes can thus provide information on the
level, availability and utilizability of endogenous energy sources and on the ability
of the cells to utilize exogenous substrates. Our results with y e a s t subjected to
prolonged aerobic starvation, action of inhibitors, elevated t e m p e r a t u r e or lyophilization show t h a t the combination of spontaneous and substrate-induced H+ extrusion,
the so-called acidification power, can be used as a sensitive indicator of the overall
metabolic state of the cells (Sigler et al. 1982).
MATERIALS AND METHODS
Yeast strains. Aneuploid strain Saccharomyces cerevisiae K (CCY 21-4-60) was
maintained on malt agar slants and grown in a synthetic medium as described b y
Sigler et al. (1981a) for 20 h at 30 ~ The harvested cells were washed three times
with sterile distilled water (5-rain centrifugation at 3000g). This washing s t e p is
i m p o r t a n t since otherwise the subsequent changes in H+ extrusion are o b s c u r e d
396
M. OPEKAROVA and K. SIGLER
Vol. 27
9
pH
8
7
6
20 1
\
\
FIG. 1. Time course of spontaneous ( t o p ) and
substrate-indueed ( b o t t o m ) pH changes in
yeast suspension starved for different time
intervals. N u m b e r s a t c u r v e s - - the time of
starvation in h. Initial suspension density
30 mg/mL. Glucose concentration ( b o t t o m )
45 raM; a r r o w s - - glucose additions.
by remnants of growth m e d i u m ' a t the cell surface. Experiments with lyophilized
yeast were done on commercial dried yeast (Slovlik, TrenSin, Czechoslovakia).
Starvation. Washed cells were suspended in sterile distilled water (dry mass 15 to
30 mg/mL, suspension volume 200 mL) in 2000-mL sterile cultivation flasks and
incubated at 30 ~ on a reciprocal shaker. At intervals 20-mL samples were taken,
their p H was measured and NH8 concentration and buffering capacity were determined in the s u p e r n a t a n t (centrifugation, 3000g, 5 rain). The cell pellet was resuspended in 20 m L distilled water (pH adjusted to 6.0--6.3) under constant p H
recording to determine the acidification power.
I n some experiments washed cells (13.5 mg/mL) were incubated with 100 FM
N-ethylmaleimide (Koch-Light) or 1 ~ antimyein A (Sigma) for 15 min or exposed
to 40 ~ and then starved as above.
Acidification power. Three-times washed cells were transferred into fresh water
(pHinit 6.3) at 30 ~ and incubated for 10 min; the suspension p H at the end of
this interval was denoted pHz0. Glucose was then added to a concentration of
45 mM and the suspension p H was measured after another 10 min (pH20). The
difference between the initial and the final p H gives the acidification power AP:
AP ----- (pHinft - - pHzo) + (pHzo -- pH2o) :
pHlntt -- pg2o 9
1982
A C I D I F I C A T I O N P O W E R OF S. cerevisiae
391
Although the pill0 is cancelled out in the equation, the 10-rain incubation before
glucose addition is crucial since it allows the cells to attain a spontaneous pH level
characteristic for their metabolic state. The 10-min interval for both spontaneous
and substrate-induced pH change was chosen because in most cases the changes
were completed within it; longer intervals make the measurement time-consuming,
especially with a higher number of samples.
The effect of external alkalinity on the magnitude of H + extrusion was determined
in cells taken from a 20-h suspension, washed twice on a Synpor 4 membrane filter
......
9
:
. . . . . . . . . .
I
-
J
"
3
ApH
2
1
0
-I
zz
-
-2L
q
-I
-3~10 - l o p
.~3
N H 3 Io
]150
;ATP 1 02
i
i
I
BC
~100
I
E
-1,1
5o
[
i
1
i
0
4-0
80
--'~1
120
--
0
FIG. 2. Top: Effect of s t a r v a t i o n on spontaneous p H change (closed squares), glucose-induced p H change
(open squares) a n d total acidification power. Initial suspension density 30 mg dry mass per mL. Acidification
power curve was obtained b y the least squares m e t h o d from three experiments (circles); p H change curves
correspond to experiment signed b y closed circles. I, I I -- segments of A P curve corresponding to equations
(3) a n d (4). Bottom: Effect of s t a r v a t i o n on :NHs level in starving yeast suspension (retool/L, curve 1), its
buffering capacity (BC, ptmol H + per L per p H unit, curve 2), A T P level in cells (p~gper mg dry mass, curve 3)
a n d initial 02 consumption rate b y the cells (nmol per m i n per m g dry mass, curve d). Initial suspension
density 17 m g dry mass per mL. All values are means of 2 determinations.
398 M. OPEKAROVAand K. SIGLER
Vo]. 27
TABLEI. Effect of starvation on the pH and the bufferingcapacity of cell-freeextract
Time of
starvationa
h
pH of cell-free
extract
Bufferingcapacity
y~rnolH + per mg protein per
pH unit
2
23
48
72
6.97
6.93
7.68
7.91
29.7
13.1
4.2
4.8
aInitial values (no starvation) cannot be obtained as the preparation of the extract takes about 2 h.
and suspended immediately in water of given p H adjusted with NaOH. Both types
of acidification were measured for 10 min as described above.
The n u m b e r of viable cells was determined just before and 30 rain after glucose
addition by vital staining with methylene blue (Arnold 1973). The cell d r y mass was
estimated by filtering 2-mL suspension samples through a Synpor 4 filter, rinsing
the pellet with 3 • 1 m L water to remove remnants of lyzed cells, transferring the
pellet q u a n t i t a t i v e l y to aluminium counting plates and weighing it after overnight
drying at 105 ~
NHa concentration in suspensions was determined according to Conway (1957)
and the buffer power b y titration with 15 mM H~S04 or 100 mM N a O H to p H 7. The
rate of oxygen consumption was measured in a Clark-type oxygen electrode at a cell
concentration of 0.24 mg/mL. A T P was extracted from the cells b y boiling ethanolic
glycine buffer (pH 9.0) for 5 rain (Kalbhen and Koch 1967) and assayed according to
McElroy (1963) using the lucipherin--lucipherase system (Serva) on a B e c k m a n
LS 9000 scintillation counter at room temperature.
Cell-free extract was prepared b y disrupting the cells in a homogenizer with glass
beads at a 1 : 1 volume ratio. The homogenate was decanted and centrifuged at
10 0 0 0 g for 10 min and at 100 0 0 0 g for 90 rain. The protein content was assayed
according to L o w r y et al. (1951). The buffer power of the extract and the titratable
acidity produced therein by glucose fermentation were determined b y supplying
2 m L extract (protein concentration 1.8 -- 2.5 mg/mL) with 0.1 m L 40 % glucose
and incubating it (20 min, 30 ~
under constant stirring and p H monitoring.
0.1 m L 10 mM N-ethylmaleimide was then added to stop glycolysis and the e x t r a c t
was t i t r a t e d with 100 mM N a O t t (2-h and 23-h starvation) or 15 mM H2SO4 (48-h
a n d 72-h starvation) to p H 7. The acidity determined from t i t r a n t consumption was
compared with t h a t obtained in a glucose-free control. The latter value served also
for determining the buffering capacity.
RESULTS
Spontaneous and substrate-induced p H change, autolysis, energy level
The spontaneous H+ extrusion observed on transfer of yeast cells into water or
an unbuffered medium (Fig. 1 top) was pronounced in fresh cells and its magnitude
decreased with proceeding aerobic starvation. I n contrast, the substrate-induced H +
efflux observed on addition of glucose to the resulting yeast suspension (e.g. Riemersma and Alsbach 1974; Sigler et al. 1981a, b) increased in the first 24 h of starvation
and t h e n declined (Fig. 1 bottom). Both types of acidification were superimposed on
another p H change; cells starved aerobically in water for 1--2 h and transferred
into fresh water caused its alkalization which became more pronounced as the
1982
ACIDIFICATION
P O W E R O F S. cerevisiae
:399
TABLE I I . E f f e c t o f s t a r v a t i o n o n t h e t i t r a t a b l e a c i d i t y o f cell-free e x t r a c t (CFE) a n d on a c i d i t y p r o d u c t i o n
d u e to glucose f e r m e n t a t i o n
Titratable acidity
~mol H + per mg protein
T i m e of
starvation
ApHCFE d u e to
glucose
fermantation
CFE
C F E ~- glucose
n e t glucose-produced
2
23
48
72
0.02
0.03
0.05
0.04
0.89
0.92
2.85 a
4.35 a
1.97
2.04
2.19 a
3.85 a
1.08
1.12
0.66
0.50
aThe v a l u e s d e n o t e a c t u a l l y t h e t i t r a t a b l e a l k a l i n i t y o f cell-free e x t r a c t f r o m e x t e n s i v e l y s t a r v e d cells;
g l u c o s e - p r o d u c e d a c i d i t y is t h e n d e m o n s t r a t e d as a decrease in t h e a l k a l i n i t y .
starvation proceeded. Accordingly, the p H of starving y e a s t ~uspensions rose steadily
to 8 - - 9 after some 24 h of starvation. This alkalization was found to be due to
ammonia produced apparently in autolytic reactions occurring in cells deprived of
exogenous carbon and nitrogen sources. The level of ammonia in suspension filtrates
increased to a b o u t l0 mmol/L after 120 h of starvation (Fig. 2 bottom). The buffering
capacity of suspension filtrates also increased, apparently due to the presence of
ammonia and other buffering substances released from the cells (Fig. 2 bottom).
The starvation caused a linear decrease in suspension d r y weight according to the
equation
dwt = 100 -- 0.112 t
(1)
where dwt is suspension dry mass in per cent after t hours of starvation, 100 is initial
d r y mass (100 ~/o) and the coefficient 0.112 is the relative d r y mass decrease per 1 h
(mean from 4 experiments, SE --~ 0.01). The proportion of dead cells in the suspension was 2 - - 8 ~/o at all starvation times up to 120 h when it increased sharply
to 25 ~/o or more.
Other changes observed during the starvation included a decrease in the cellular
level of A T P and in the initial rate of oxygen consumption b y cells transferred from
starving suspension into an oxygen electrode chamber (Fig. 2 bottom). Both the A T P
level and the oxygen consumption rate registered a sharp drop in the initial phases
of starvation and a slower decrease at further intervals. The rate of oxygen consumption measured with the oxygen electrode was found to be higher than the
endogenous respiration rates measured conventionally b y the W a r b u r g technique.
The difference m a y be due to the fact t h a t manometric measurements are usually
done for 1 h or more (with an initial 5 - - 1 0 rain temperature equilibration interval)
under conditions much like those in a starving suspension while our oxygen electrode
measurements were performed for the initial 5 - - 7 rain. T h e y thus reflected the level
of endogenous substrates available for respiration immediately on transfer of the cells
into a fresh medium.
Acidity and buffering capacity of cell-free extract
As shown in Table I the p H of cell-free extract rose with proceeding starvation
while its buffering capacity decreased (its slight increase after 3 d of starvation m a y
be due to accelerated autolysis producing buffering substances). Table I I shows that,
owing to the high buffering capacity of the extract (cf. Sigler et al. 1981b), glucose
fermentation produced hardly any p H change although it gave rise to considerable
400
M. OPEKAROV/~ and K. S I G L E R
Vol. 27
titratable acidity. With proceeding starvation this acidity also decreased, mirroring
the decreasing glycolytic capacity of the extract. The decline in cell homeostasis
seems to be best reflected in the drop in intracellular buffering capacity (Table I).
Acidification power
In Fig. 2 (top) spontaneous and substrate-induced p H changes are plotted against
the time of starvation. Acidification is t a k e n as positive, alkalization as negative.
0.6
!
I
I
rnmo[/L
0.5
t5
~urnoi/L
O./-,.
!
1
43
0,3
0.2
0.1
I
0
10
[. . . . . . . .
20
~
30
mg/mL
FI(~. 3. Effect of suspension density on the magnitude of spontaneous (closed symbols, ~zmol/L) and substrateinduced (open symbols, mraol/L) H + extrusion in S. cerevislae.
The sum of the two values, the acidification power A P (cf. Methods), decreased
linearly with the time of starvation:
APt -----Al:)init - - Bt
(2)
where APinit is the initial A P value in fresh cells, B is its change per 1 h of starvation
and t is time of starvation in h. APinit and B are characteristic values for given y e a s t
b a t c h and growth conditions. In our experiments the following equation held for
starvation intervals up to 70 h:
APt -~ 2.47 -- 0.029 t
(3)
with a determination coefficient r 2 ---- 0.953 (3 experiments). On further starvation
the equation assumed the form
APt : 4.81 -- 0.062 t (r 2 -~ 0.930, 3 experiments)
(4)
Cells with A P of 2.5 to 1.0 are seen to be in a good physiological state, those with
A P of 1.0 to 0 are partially damaged and cells with A P < 0 are considerably damaged.
The extent of acidification, and thus the AP, depend on cell concentration (Fig. 3).
Reproducible A P values are thus obtained at cell concentrations above 10 mg/mL;
A C I D I F I C A T I O N I~OWER OF kq. cerevisiae
1982
T
l
40|
I
ApH
"2
0
l
I
10
2O
30
FIG. 4. Changes in acidification power caused
b y different t r e a t m e n t s ; ? l ~ a n t i m y e i n A;
2 100 ~
N-ethylmaleimide; 3 g r o w t h a t
30 ~ s t a r v a t i o n a t 40 ~ 4 growth a t 40 ~
s t a r v a t i o n at 30 ~ 5 revival a n d s t a r v a t i o n
of commercial dried y e a s t at 30 ~ 6 control
cells grown a n d s t a r v e d a t 30 ~
Initial
suspension d e n s i t y 13.5 m g d r y m a s s per m L .
otherwise a constant cell concentration has to be maintained throughout the experiment.
A n y change in growth conditions or t r e a t m e n t with inhibitors causes changes in
APin~t (short-term effect) or in coefficient B (long-term effect) or both (Fig. 4). Short
t r e a t m e n t with N-ethylmaleimide had a short-term effect which persisted to a b o u t
the same extent for the following 30-h starvation. In contrast, antimycin A prevented
the normal A P drop on further starvation. This effect m a y reflect energy dependence
of protein turnover; if protein degradation required energy (cf. Wolf 1980) in the
form of A T P and if antimycin A blocked A T P formation, then the normal starvationinduced degradation of membrane proteins participating in H + extrusion would be
slowed down or abolished and acidification power would not be affected b y starvation.
This possibility is supported b y the results of }talvorson (1958a, b), tIemmings (1978)
and Maz6n (]978) who found an inhibition of protein degradation in yeast b y sodium
azide and 2,4-dinitrophenol.
Growth of yeast at 40 ~ with subsequent starvation at 30 ~ had a lower effect
on A P than the reverse situation, i.e. growth at 30 ~ and starvation at 40 ~ This
m~y reflect the higher protein degradation in resting cells as compared with growing
ones (Halvorson 1958a, b; Betz ]976).
To test the usefulness of A P for industrial purposes we studied the revival of commercial dried yeast on transfer into water. The A P of the resulting suspension
increased for a b o u t 30 rain when the yeast reached full metabolic activity (in keeping
with the manufacturer's guarantee). The m a x i m u m A P value was lower than t h a t
of our pure strain b u t the subsequent decrease in A P on starvation was similar.
402
M. OPEKAROV/f~ a n d K. S I G L E R
Vol. 27
DISCUSSION
Treatments which drain the cellular energy sources can be likened to aerobic
starvation of different duration which can thus be used as a reference basis for the
assessment of cell impoverishment. The acidification power, used here as an indicator
of the impoverishment, changes during the starvation in a way which makes it
a useful tool for estimating the metabolic vigour of the cells.
The effect of aerobic starvation on yeast cells is partially due to the fact that
under non-growth (starvation or differentiation) conditions the activity of yeast
pretenses increases drastically as a part of the adaptation of the cells to poorer conditions (Wolf and Holzer 1980; Wolf 1980). The ensuing amino acids are deaminated
to serve in anaplerotic reactions as intermediates of the citric acid cycle and may
serve as a source of the ammonia found in starving yeast suspensions. The NH8
production is probably responsible for the apparent H + uptake observed in yeast
suspensions starved for 1 d or more (Rodriguez-Navarro and Sancho 1979; Ps
1981). A 1-d starvation under our conditions caused marked changes in cell substrate
and energy levels, cell homeostasis, and the function of membrane transport systems.
Although under suitable storage conditions the changes will be much smaller, our
results indicate that data obtained with yeast cultures stored or starved for 1 d or
more should be interpreted with caution and care should be taken to determine
their acidification power (relative to fresh cells) or some other parameter indicating
the metabolic condition of the cells.
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