Protective Role of Trehalose in Thermal Denaturation of
Yeast Pyrophosphatase
M au ro S ola-P enna and Jose R o b erto M eyer-Fernandes
D epartam ento de Bioquimica Medica, Instituto de Ciencias Biomedicas, Universidade Feder­
al do Rio de Janeiro, 21941-590, Rio de Janeiro, Brasil
Z. Naturforsch. 49c, 327-330 (1994); received January 7, 1994
Thermal D enaturation, Trehalose, Yeast Pyrophosphatase, W ater Activity, Carbohydrates
Trehalose, a disaccharide o f glucose, is accumulated in yeast cytosol when this organism is
submitted to a stress condition. Recently it was shown that the level of trehalose increase up to
15 times when yeast cells are submitted to heat shock (De Virgilio et al., 1991). In this report
we give evidence how trehalose may play an im portant role on the stress-survival o f yeasts
when submitted to a heat shock. We show that 1.5 M trehalose increases 13-fold the half-time
for thermal inactivation ( / 0 5) o f yeast cytosolic pyrophosphatase at 50 °C. This thermal pro­
tection conferred by trehalose is dose-dependent, after 10 min at 50 °C, a condition which
inactivated pyrophosphatase, the presence of 2 m trehalose preserves 95% of total activity.
O ther carbohydrates were tested but were not so effective as trehalose. The presence o f tre­
halose at high concentrations in the reaction medium at 35 °C inhibits pyrophosphatase activ­
ity. This inhibition is less effective at 50 °C suggesting that under this condition the enzyme is
tem perature-protected and active.
Introduction
T rehalose, a non-reducing disaccharide o f glu­
cose, is widely distrib u ted am o n g living systems
reaching high concen tratio n s in cells cytossol w hen
these organism s are under stress conditions
(C row e et al., 1984). Such high co ncentrations can
reach 35% o f dry w eight o f the cells (Crowe et al.,
1984; W iem ken, 1990), th at in h y d rated cells reach
the m olar range. T rehalose is considered to have
an im p o rta n t role in o sm oregulation and in the
ability o f organism s such as b a k e r’s yeast to su r­
vive severe d eh y d ratio n an d low -tem perature
stress (Clegg and Filosa, 1961; C row e et al., 1984;
Sussm an and L ingappa, 1959). In some cells, tre ­
halose accum ulation is induced by heat shock.
T his pheno m en o n is reversible an d trehalose is
rapidly degraded u p o n retu rn o f the cells to p h y ­
siological conditions. It has been proposed th a t in
yeast, trehalose does not prim arily function as a
storage co m p o u n d b u t as a highly efficient p ro ­
tecting agent to m ain tain the stru ctu ral integrity o f
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the cytoplasm under environm ental stress condi­
tions (De Virgilio et al., 1991; Neves et al., 1991;
W iemken, 1990).
We have show n th a t carb o h y d rates can m o d u ­
late the catalytic cycle o f io n -tran sp o rtin g ATPases.
They decrease the K m for Pi in the p h o sphorylation
reaction catalyzed by (C a2+ + M g2+)A T Pase from
sarcoplasm ic reticulum (Chini et al., 1991) an d in ­
crease the rate o f ATP-e* Pi exchange catalyzed by
(C a2+ + M g2+)A TPase from renal plasm a m em ­
branes (Vieyra et al., 1989, 1991). B oth reactions
are stim ulated by a decrease in w ater activity and
in protein solvation (C hini et al., 1991; de Meis,
1989). Recently it has been show n th a t a decrease
in protein solvation pro m o ted by sugars can regu­
late the equilibria am ong allosteric conform ations
o f hem oglobin (C olom bo et al., 1992). These d ata
indicate th a t the flexibility and dynam ic n atu re o f
proteins are affected by pro tein -b o u n d w ater m o ­
lecules, in ways th a t can be m odified by interaction
w ith biological solutes. A t high tem peratures, c ar­
bohydrates can stabilize protein structure by driv­
ing the equilibrium betw een native and unfolding
configurations in the direction o f the native state.
The present re p o rt exam ines the role o f treh a­
lose in therm al d en a tu ra tio n and catalytic activity
o f yeast cytosolic pyro p h o sp h atase and suggest
why trehalose b ut n o t o th er carb o h y d rates are ac­
cum ulated by yeast u n d e r stress conditions.
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328
M . Sola-P enna an d J. R. M ey er-F ernandes • T rehalose-Induced T herm al S tability o f Y easts P y ro p h o sp h atase
Materials and Methods
Y east inorganic p y ro p h o sp h atase, trehalose and
tris were purchased from Sigm a Chem ical Co.
O th er reagents were o f the higher pu rity available.
P y ro p h o sp h atase activity was determ ined by
m easuring the to ta l Pi released in the end o f reac­
tion.
D a ta points show the m eans o f four experi­
m ents, w ith sta n d a rd errors o f less th a n 10%. F it­
ting were done using the non-linear regression
co m p u ter p ro g ram E nzfitter (Elsevier, Biosoft).
G o odness o f fit was assessed by com puting re­
duced chi squares for the fits, as described in
(M o tu lsk y a n d R ansnas, 1987).
Results
W hen yeast cytosolic p y ro p h o sp h atase was p re­
in cubated at 50 °C its activity m easured in a stand-
ard assay m edium o f 35 °C, decreased as a func­
tion o f pre-incubation tim e. The ad d itio n o f tre­
halose to the pre-incubation m edium protected the
enzyme against inactivation, increasing its stability
at high tem perature (Fig. 1). In the absence o f tre ­
halose less than 2 0 % o f the original activity re­
m ained after pre-incubation for 25 m in a t 50 °C.
The addition o f 1.5 m trehalose to the pre-incubation m edium preserved 80% o f the original activi­
ty under the same conditions. The degree o f p ro ­
tection depended on the trehalose concentration:
after a 10 m in pre-incubation at 50 °C in the pres­
ence o f 0.4 m trehalose, h a lf o f the c o n tro l activity
w ithout pre-incubation w as retained; w ith 2 m tre ­
halose, activity was 95% o f the co n tro l value
(Fig. 2 ).
The ability to stabilize pyro p h o sp h atase at high
tem perature was specific to trehalose. Table I
shows th a t other carb o h y d rates were n o t nearly so
effective. W hereas 1.5 m trehalose increased the
half-tim e (/05) for tem p eratu re inactivation by
13-fold, the greatest effect detected w ith the other
carbohydrates used w as a 3.3-fold increase in t0 5.
A second effect, also specific for trehalose, was
also observed: the presence o f trehalose in the
Time (m in )
Fig. 1. Time course of thermal denaturation of pyro­
phosphatase at 50 °C. Yeast cytosolic pyrophosphatase
(40 |ag/ml) was pre-incubated for the times indicated in
the abscissa in the absence (O) or in the presence ( • ) of
1.5 m trehalose. After pre-incubation, 50 vols. of reac­
tion medium was added, at 35 °C. Final concentrations
in the reaction medium were 100 m M Tris/HCl pH 7.5,
10 m M MgCl2, 2 m M pyrophosphate, 0 or 30 m M trehal­
ose and 0.8 ng/ml pyrophosphatase. The reaction was
quenched after 1 min by addition of 2 vols of 2 0 % (w/v)
trichloroacetic acid. After dilution, the activity (100%
on the ordinate) was 615 ± 50 nmol Pi •m g “1 •m in “1 with
or without 30 m M trehalose. The data were fitted by non­
linear regression to the equation v = Vo ■e~kl, where Vo is
the initial rate of pyrophosphate hydrolysis without pre­
incubation; k is the decay constant; t is the pre-incubation time.
0 .0
0 .5
1.0
1.5
2 .0
[Tre ha lose] (M)
Fig. 2. Trehalose concentration dependence for protec­
tion against thermal denaturation. (O) The enzyme was
pre-incubated for 10 min at 50 °C in the presence of the
concentrations of trehalose indicated on the abscissa.
Reaction was started as in Fig. 1, and the trehalose was
diluted 50-fold by addition o f reaction medium, at
35 C. ( • ) Reaction measured without pre-incubation.
Other conditions as in Fig. 1.
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M . S ola-P enna an d J. R. M ey er-F ern an d es • T rehalose-Induced T herm al Stability o f Y easts P y ro p h o sp h a ta se
Table I. Thermal denaturation of pyrophosphatase in
the absence and presence of different carbohydrates. The
enzyme was pre-incubated for different times at 50 °C in
the presence of the indicated concentrations of carbohy­
drates, then diluted in the reaction medium and tested
for pyrophosphatase activity as described in Fig. 1. The
fit and the equation used were the same as in Fig. 1. /0s
represents the half-time for loss o f pyrophosphatase
activity.
Carbohydrate present
during pre-incubation
C oncentration
None
Glucose
Fructose
Sucrose
Trehalose
_
[M ]
3
3
1.5
1.5
?0.5
[min]
5.2 ± 0.5
8.5 ± 0 .9
1 2 . 8 ± 1.1
16.9 ± 1.4
60.3 ± 0.8
reaction m edium inhibited the enzym atic activity
o f pyro p h o sp h atase (Fig. 3). A t 35 °C, 2 m tre ­
halose reduced the activity to 1 0 % o f the control
value. O th er carb o h y d rates were less inhibitory:
w ith the sam e co n cen tratio n o f sucrose (o r twice
the co n cen tratio n o f glucose an d fructose), 75% o f
the control activity rem ained (Fig. 3 A). W hen the
reaction w as carried o u t a t 50 °C, trehalose inhib­
ited less th a n a t 35 °C (Fig. 3 B), w hereas the oth er
carb o h y d rates h ad the sam e effect as before.
Discussion
The ability o f carb o h y d rates to stabilize p ro ­
teins has been a ttrib u te d to th e preferential h y d ra­
tion o f p ro tein th a t occurs w hen it is dissolved in a
carb o h y d rate solution (A rak aw a and Tim asheff,
1982, 1983; Lee an d T im asheff, 1981). This p he­
o
c
o
(j
o
[ D i s a c c h a r i d e ] (M)
0.0
1.0
[ M o n o s a c c h a r i d e ] (M)
329
nom enon is due to the fact th a t polyols, in general,
and unlike destabilizing agents such as guanidinium salts (A rakaw a and Tim asheff, 1984), solu­
bilize in bulk w ater. A n increase in the co n cen tra­
tion o f carb o h y d rate in bulk w ater engages m ore
w ater m olecules in solubilization and a t high ca r­
bohydrate concentrations, protein an d solutes be­
gin to com pete for the available w ater. This com ­
petition leads to a reduction in the p ro tein solva­
tion layer, w hich results in a decrease in protein
apparent m olal volum e (A rakaw a an d Tim asheff,
1982). As a result, the p ro tein becom es m ore com ­
pact and less susceptible to the destabilizing forces
o f high tem perature (Back et al., 1979).
Preferential hy d ratio n studies have been done
w ith several carb o h y d rates b ut n ot trehalose (A ra­
kaw a and Tim asheff, 1982). T he specificity o f the
effects o f trehalose show n in this study suggest
th at this carb o h y d rate at high co n cen tratio n can
interact directly w ith the protein. It m ay be th a t
this interaction stabilizes py ro p h o sp h atase fo rm ­
ing therm odynam ically stable structures in such
way th at the resulting con fo rm atio n has a low en­
zym atic activity (K libanov, 1983). In h ib itio n o f
enzyme catalysis by trehalose was m ore effective at
35 °C th an when activity was m easured at 50 °C
(Fig. 3), suggesting th a t a t the higher tem perature,
increased protein m obility p ro m o ted recovery o f
the catalytic activity o f this enzyme.
The results show n here are consistent w ith the
idea th at preferential h y d ra tio n is n o t the only fac­
to r in stabilizing the native structure o f proteins
(A rakaw a and Tim asheff, 1985; A rak aw a et al.,
1990; Lee and Lee, 1987). The m agnitude o f the
stabilizing effect is show n here to depend on the
Fig. 3. Effects of different mono- and disac­
charides on pyrophosphatase activity. (A)
Reaction at 35 °C. The activity in the absence
of sugars was 613 ± 48 nm ol-m g “1 m in-1. (B)
Reaction at 50 °C. The activity in the absence
o f sugars was 942± 80 nm ol-m g “1 ■min"1. The
assay medium was the same as in Fig. 1. Reac­
tion was started by addition of soluble yeast
pyrophosphatase to a final concentration of
0.8 ng/ml, and concentrations of trehalose (O),
sucrose ( • ) , glucose (□ ) and fructose (■ ) are
indicated on the abscissa. The reaction was
quenched after 1 min, and pyrophosphatase
activity was determined as in Fig. 1.
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330 M . S ola-P enna an d J. R. M eyer-F ernandes • T rehalose-Induced T herm al Stability o f Y easts P y ro p h o sp h atase
n atu re o f the sugar, and m ay also depend on the
n atu re o f the protein . The ability to p ro m o te th e r­
m al stability and inhibit p y ro p h o sp h atase activity
m ay be related to the different physical properties
o f the carb o h y d rates tested (D u d a and Stevens,
1990).
T he study o f organism s th a t have ad ap ted to
conditio n s o f d eh y d ratio n , h eat, o r high osm otic
activity provides a clue to how n atu re has a p ­
pro ach ed the p roblem o f m ain tain in g a functional,
viable organism u n d er conditio n s th a t m ight nor-
mally be expected to d en atu re o r inactivate a large
num ber o f th a t organism s m acrom olecules. The
specific effectiveness o f trehalose in protecting
against therm al d en atu ratio n o f yeast p y ro p h o s­
phatase m ay explain why these cells selectively ac­
cum ulate trehalose and n o t o th er carbohydrates.
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A cknow ledgem en ts
We th an k D r. M arth a M . Sorenson for the dis­
cussions a b o u t this m anuscript.
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