AbstractThe rate of heat production in a periodically glycolysing cell-free cytoplasmic medium extracted from yeast Saccharomyces cerevisiae is measured with a batch calorimeter. The rate exhibits periodic variations of approx. 10% of the average heat production rate of about 54 mJml per minute. From this rate and the enthalpy change fro glycolysis a glucose degradation rate of 0.43 mMming is calculated. The value fits into the ‘oscillatory window’ determined by a glucose injection technique

Hess, B.

Lamprecht, I.

Müller, S.C.

Plesser, T.

Schaarschmidt, B.

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Periodic heat production by oscillating glycolysis in a cytoplasmic medium extracted from yeast 

Volume 189, number 1 FEBS 2861 September 1985 
Periodic heat production by oscillating lycolysis in a 
cytoplasmic medium extracted from yeast 
T. Plesser, S.C. Mtiller, B. Hess, I. Lamprecht* and B. Schaarschmidt’ 
Tfie rate of heat production in a periodically glywlysing cell-free cytoplasmic medium extracted from yeast 
Saccharomyces cerev&iae is measured with a batch calorimeter. The rate exhibits periodic variations of 
approx. 10% of the average heat production rate of about 54 mJ/ml per minute. From this rate and the 
enthalpy change fro glycolysis a glucose degradation rate of 0.43 mM/ming is calculated. The value fits 
into the ‘oscillatory window’ determined by a glucose injection technique. 
Glycolysb Microcalorimetry Heat production Enthalpy 
1 e INT~OR~CTIUN 
The production of heat and its regulation ac- 
companied by metabolic turnover is an inherent 
property of living systems. Therefore calorimetry 
is an adequate tool for the analysis of dynamic 
processes in biology and biochemistry. Calori- 
metric measurements have clarified the relation 
between growth and heat production of biological 
systems, On the other hand, they have been helpful 
for the elucidation of elementary reaction and 
binding mechanisms in chemical as well as bio- 
chemical problems flf. Much less is known about 
heat production by distinct metabolic pathways 
and their enzyme catalysed reaction steps. An 
obvious candidate for such an investigation is the 
highly exothermic anaerobic degradation of sugar 
by the sequence of glycolytic reactions. Examples 
of calorimetric research work on glycolysis in in- 
tact cells can be found in the literature [l-3]. 
Glycolysis in a celtfree cytoplasm may be con- 
sidered as a better defined model system since 
other metabolic pathways are excluded and sub- 
i%dicated to Professor Cart Martins on the occasion of 
his 80th birthday 
strates as well as inhibitors specific for different 
reaction steps can be supplied in a precisely con- 
trolled manner 141. 
In this paper a study of heat production during 
oscillating glycolysis is presented to determine the 
heat balance of this particular dynamic state of the 
glucose metabolism. In addition, the study was 
motivated by the pattern formation process in thin 
layers of cytoplasm from yeast first reported by 
Boiteux and Hess [5]. These patterns, observed at 
NADH-specific wavelengths, arise and disappear 
twice within one NADH cycle during those time in- 
tervals in which the NADH concentration changes 
at its maximum rate on the ascending or descend- 
ing part of the cycle, respectively. The pattern is 
connected to convective flow driven by gradients in 
surface tension [6]. Since the surface tension 
depends on temperature, and heat may be pro- 
duced when the 2 different parts of the glycolytic 
pathway are activated, one can speculate that the 
production of heat in a periodically metabolizing 
extract has half the period of the photometrically 
monitored NADH oscillations. 
The experiments how for the first time oscilfa- 
tions of the heat produ&tion in a cell-free glycolytie 
system, The period of the oscillations is the same 
in calorimetric and photometric measurements. 
42 
Published by Eisevier Science Publishers E. V. (Bmmedrral Division) 
00145793/85/$3.30 @ 1985 Federation of European Biochemical Societies 
Volume 189, number 1 FEBS LETTERS September 1985 
2. MATERIALS AND METHODS 
The cytoplasmic medium used for the ex- 
periments was extracted from yeast cells (Sac- 
c~aro~yces Ca~ls~erge~s~) grown under aerobic 
conditions according to a published procedure [7]. 
The protein content of 47 mg/ml corresponds to 
roughly one third of the concentration in the 
cytoplasm of intact cells. The procedure of sample 
preparation was the same for photometric and 
calorimetric experiments. For calorimetry the sam- 
ple volumes are increased by a factor of 6 com- 
pared with the following volumes used for 
photometry: 300~1 extract; 40 /cl of 0.7 M 
trehalose dissolved in 0.1 M potassium phosphate 
buffer; 10 ~1 of 20 mM NAD; 30~1 of 1 M 
potassium phosphate. The samples were thorough- 
ly mixed before use. This recipe leads to oscillatory 
fermentation activity in the extract. All chemicals 
were of analytical grade. 
Spectrophotometric measurements were done 
with a 2 mm quartz cuvette in a Zeiss PM4 spec- 
trophotometer. Calorimetric me~urements were 
performed with a microcalorimeter E. Calvet 
(SETARAM, Lyon) with 2 pairs of vessels allow- 
ing for 2 simultaneous experiments. In each pair 
one vessel served as the reaction chamber, the 
other as a reference. Their sensitivity amounts to 
63.1 and 60.3 ~~/mW, respectiveiy. The vessels 
with a volume of 15 ml could be equipped with 
stirrers. For a technical description of the instru- 
ment see [8,9]. 
Preparing the experiment, samples of approx. 
2 ml were filled into the reaction chambers. The 
reference chambers received the same volume of 
water. The top of each calorimeter vessel carried a 
small cup of 1 ml with a removable bottom. For 
titration experiments the titrand was placed into 
these cups and released, at an appropriate point in 
time, into the sample by pushing down the bottom. 
The temperature of the calorimeter was kept at 
275°C. No signal corrections were made for the 
time constant of the calorimeter which is less than 
2 min for a sample volume of about 2 ml. 
3. RESULTS 
Fig. 1 shows the periodic rate of heat production 
4 by a sample of 2.3 ml yeast extract without stir- 
ring. The difference between maximum and 
minimum rate during one period is 0.2 mW 
(0.087 mW/ml). Thus the periodic variation 
modulates the slowly decreasing overall rate 4 SI: 
2 mW (0.87 mW/ml) by approx. 10%. The period 
of the oscillations is I2 min. This value is close to 
the period of 13 min which was measured with a 
380 pl sample in a photometer at a NADH specific 
wavelength (fig.2). 
The wave form of the oscillations in fig.1 has 
some characteristic features as for example the 
sharp rise after the mi~mum and its buckhng as- 
cend to the maximum. The descend from the max- 
imum shows a smoother profile. The shape differs 
significantly from that of the curve in fig.2 
Fig. 1. Oscillations of 4, the rate of heat produced by a 2.3 ml sample of cell-free extract of the yeast S. Cdsbergensis 
fed with trehalose. The difference between minimum and maximum 4 amounts to 2 mW (0.087 mW/ml). The period 
is 12 min. 
43 
Volume 189, number 1 FEBS LETTERS September 1985 
Fig.2. Oscillations of NADH concentration monitored photometrically at 340 nm in a sample of cell-free yeast extract 
from the same preparation as in fig.1 and at the same temperature within f 05°C. The period is 13 min. 
representing the time course of the concentration 
of NAD or NADH, respectively. Running the 
calorimeter with permanent stirring of the samples 
did not alter the results. 
4. DISCUSSION 
A consideration of the enthalpy change of the 
overall reaction [3,1 l] 
Trehalose + Hz0 ---+ 2 glucose 
AHSO 
Glucose + 2 CO2 + 2 ethanol 
AH = - 126 kJ/M 
allows the calculation of the glucose turnover, 
which results in a rate of 0.43 mMfm1. This value 
fits into the ‘oscillatory window’ ranging from 0.3 
to 2 mM/min as determined by a glucose injection 
technique [4, lo]. 
The splitting of fructose 1 ,dbisphosphate by the 
enzyme aldolase and the reduction of acetaldehyde 
by the enzyme alcohol dehydrogenase, the last step 
in the fermentation process, should be considered 
as the major sources for the varying rate of heat 
production. The aldolase reaction is endothermic 
with AH = + 42 kJ/mol glucose [2]. The produc- 
tion of ethanol from acetaldehyde is accompanied 
by a release of heat in the range of AH = 
- 47 kJ/mol 1121 or AH = - 94 kJ/mol glucose. 
Since preliminary titration experiments with 
AMP and ADP do not show right away the pro- 
nounced phase jumps as in s~trophotometric ex- 
periments [lo], the phase correlation of heat pro- 
duction and NADH concentration has not yet been 
established. Further experiments are in progress 
44 
which, by simultaneous measurements of NADH 
concentration and heat production in one sample 
cuvette, will allow for a better identification of the 
most significant heat producing and heat consum- 
ing steps in oscillating glycolysis. 
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Periodic heat production by oscillating glycolysis in a cytoplasmic medium extracted from yeast

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