Tài liệu Báo cáo khoa học: Temperature and phosphate effects on allosteric phenomena of phosphofructokinase from a hibernating ground squirrel (Spermophilus lateralis) pptx

Temperature and phosphate effects on allosteric
phenomena of phosphofructokinase from a hibernating
ground squirrel (Spermophilus lateralis)
Justin A. MacDonald
1
and Kenneth B. Storey
2
1 Department of Biochemistry & Molecular Biology, University of Calgary, AB, Canada
2 Institute of Biochemistry and Department of Biology, Carleton University Ottawa, ON, Canada
Environments with widely differing seasonal tempera-
tures present thermoregulatory challenges to small
mammals who aim to maintain a constant body tem-
perature of about 37 °C. Winter is particularly difficult
because energy use in support of homeothermy increa-
ses dramatically in cold weather at the same time as
the food supply declines. For many small mammals,
the only survival solution to this combination of low
food availability and low environmental temperatures
is hibernation [1–3]. The mammalian hibernator aban-
dons homeothermy and allows body temperature to
drop to that of its surroundings (although regulating
body temperature at 0–5 °C if ambient temperature
falls below 0 °C). The mechanisms that control the
entry into hibernation are still not fully understood
but it is known that an active suppression of basal
metabolic rate occurs (often to only 1–5% of the nor-
mal resting rate), preceding and causing the fall in
body temperature. Hibernation is also facilitated by
the accumulation, during late summer feeding, of huge
reserves of lipids; for example, in ground squirrels,
body mass often increases by 50% or more. These

lipids are the main fuel for winter energy metabolism
during torpor and measurements of respiratory quo-
tients confirm this. Lipid oxidation is supplemented to
some extent by gluconeogenesis from amino acids but
carbohydrate reserves are largely spared to be used
only by tissues and organs that can oxidize little else
Keywords
glycolysis; mammalian hibernation;
metabolic rate depression;
phosphofructokinase; temperature effects
Correspondence
K. B. Storey, Institute of Biochemistry and
Department of Biology, Carleton University,
1125 Colonel By Drive, Ottawa, ON,
K1S 5B6 Canada
E-mail:
(Received 7 July 2004, revised 31 August
2004, accepted 14 September 2004)
doi:10.1111/j.1432-1033.2004.04388.x
Temperature effects on the kinetic properties of phosphofructokinase
(PFK) purified from skeletal muscle of the golden-mantled ground squirrel,
Spermophilus lateralis, were examined at 37 °C and 5 °C, values character-
istic of body temperatures in euthermia vs. hibernation. The enzyme
showed reduced sensitivity to all activators at 5 °C, the K
a
values for
AMP, ADP, NH
4
+
and F2,6P

2
were 3–11-fold higher at 5 °C than at
37 °C. Inhibition by citrate was not affected whereas phosphoenolpyruvate,
ATP and urea became more potent inhibitors at low temperature. While
typically considered an activator of PFK activity, inorganic phosphate per-
formed as an inhibitor at 5 °C. Decreasing temperature alone causes the
actions of inorganic phosphate to change from activation to inhibition. We
found that K
m
values for ATP remained constant while V
max
dropped sig-
nificantly upon the addition of phosphate. Phosphate inhibition at 5 °C
was noncompetitive with respect to ATP and the K
i
was 0.15 ± 0.01 mm
(n ¼ 4). The results indicate that PFK is less likely to be activated in cold
torpid muscle; PFK is less sensitive to changing adenylate levels at the low
temperatures characteristic of torpor, and PFK is clearly much less sensi-
tive to biosynthetic signals. All of these characteristics of hibernator PFK
would serve to reduce glycolytic rate and help to preserve carbohydrate
reserves during torpor.
Abbreviations
PFK, 6-phosphofructo-1-kinase; F6P, fructose 6-phosphate; F2,6P
2
, fructose 2,6-bisphosphate.
120 FEBS Journal 272 (2005) 120–128 ª 2004 FEBS
[2–4]. Glycolytic rate drops to low levels in most
organs.
Mechanisms that contribute to the suppression of

glycolytic flux are important for hibernation for several
reasons. Glycolytic rate suppression (a) contributes to
the overall metabolic rate depression by suppressing
ATP output from carbohydrate catabolism; (b) limits
carbohydrate use for anabolic purposes during torpor,
and (c) facilitates lipid oxidation as the primary ATP-
generating pathway in most organs as well as gluconeo-
genesis in selected organs. 6-Phosphofructo-1-kinase
(PFK) is an enzyme of central importance to the regu-
lation of glycolysis. PFK gates the commitment of hex-
ose phosphates (derived from glycogen or glucose) into
the triose phosphate portion of glycolysis. A wide vari-
ety of regulatory mechanisms modulate PFK activity
including allosteric control by powerful activators and
inhibitors [5], pH effects [6–8], post-translational modi-
fication by reversible protein phosphorylation [9], and
enzyme binding to subcellular macromolecules [10,11].
However, the specific mechanisms that achieve inhibi-
tion of PFK activity during hibernation have yet to be
fully realized. Skeletal muscle PFK does not appear to
be regulated by protein phosphorylation during hiber-
nation in ground squirrels [12]. As respiratory acidosis
develops during hibernation, effects of low pH on
PFK activity and subunit assembly have been sugges-
ted as a means by which glycolytic flux in skeletal
muscle can be reduced, as the enzyme is highly sensi-
tive to pH change [10,13]. However, recent work has
shown that low pH inhibition of PFK may not be
physiologically relevant [14]. Temperature-dependent
mechanisms of enzyme control may contribute to the

regulation of PFK activity as the body temperature of
hibernators can drop by 30 °C or more [15,16]. Several
examples of temperature-dependent changes in the kin-
etic properties of hibernator enzymes have been repor-
ted [16]. The present study analyzes the kinetic and
regulatory properties of PFK purified from skeletal
muscle of the golden-mantled ground squirrel (Spermo-
philus lateralis). Particular attention is paid to the
effects of temperature on enzyme allostery and the
influence of temperature in the regulation of PFK
activity at both high (euthermic) and low (hibernating)
temperatures.
Results
Selected kinetic properties of PFK purified from S. lat-
eralis muscle were compared at assay temperatures and
pH values that mimic the hibernating (5 °C, pH 7.5)
vs. euthermic (37 °C, pH 7.2) conditions found in vivo
in S. lateralis skeletal muscle. Table 1 shows the effect
of temperature on activation coefficient (K
a
) values for
allosteric activators and the values of concentration of
the inhibitor that reduces control activity by 50% (I
50
).
The enzyme showed reduced sensitivity to all activa-
tors at 5 °C, the K
a
values for AMP, ADP, NH
4

+
,
and F2,6P
2
being 5.3-, 3.4-, 2.6- and 11.3-fold higher
at 5 °C, respectively, compared with the corresponding
37 °C values. In contrast, inhibitor constants were dif-
ferentially affected by temperature change. Inhibition
by Mg citrate was not affected whereas the sensitivity
to inhibition by phosphoenolpyruvate and urea both
increased at low temperature; I
50
values dropped to 53
and 60%, respectively, of the corresponding values at
37 °C.
Inorganic phosphate is typically an activator of
PFK, and Table 1 shows that the ground squirrel
enzyme responded to phosphate as expected at 37 °C
with a K
a
value of 2.0 ± 0.2 mm. However, inorganic
phosphate was unable to elicit an activation of PFK
activity at 5 °C; in fact, the addition of inorganic phos-
phate resulted in an inhibition of PFK activity. Fur-
ther analysis of phosphate effects on PFK at 5 °C
seemed warranted and Fig. 1 shows an anomalous
interaction between pH and phosphate effects on the
enzyme. At pH 7.0, phosphate acted as a strong acti-
vator of PFK and raised maximal activity by 5.1-fold.
The calculated K

a
for phosphate was 1.73 ± 0.13 mm
(n ¼ 3). However, increasing the pH slightly to a value
of 7.3 dramatically altered the effect of phosphate and
activation was seen only at low concentrations with a
maximal 1.7-fold activation at 2 mm phosphate. At
Table 1. Effects of temperature on the kinetic properties of skel-
etal muscle phosphofructokinase from the golden-mantled ground
squirrel, Spermophilus lateralis. Values are means ± SEM; n ¼ 4–6.
F6P concentrations were subsaturating (0.1 m
M) for K
a
and I
50
determinations. For I
50
determinations, inhibitor stock solutions
were prepared with added magnesium in a 1 : 1 molar ratio for
Mg.ATP and 2 : 1 for Mg.citrate. The pH values of assay mixtures
in imidazole–HCl buffer were adjusted at 23 °C to predetermined
values so that when the mixtures were cooled or warmed to the
desired assay temperatures, the pH at 5 °C was 7.5 and the pH at
37 °C was 7.2. NA, no activation.
Conditions 37 °C5°C
K
a
AMP (lM) 34.9 ± 2.0 184 ± 5.9
a
K
a

ADP (lM) 35.5 ± 1.0 122 ± 6.4
a
K
a
NH
4
+
(mM) 1.4 ± 0.1 3.6 ± 0.08
a
K
a
Fructose-2,6-bisphosphate (nM) 39.9 ± 2.9 449 ± 15
a
K
a
Inorganic phosphate (mM) 2.0 ± 0.2 NA
I
50
Phosphoenolpyruvate (mM) 1.2 ± 0.1 0.64 ± 0.04
a
I
50
Urea (mM) 314 ± 30 189 ± 13
b
I
50
Mg.citrate (lM) 51.7 ± 5.4 48.2 ± 2.0
a
Significantly different from the corresponding value at 37 °C,
P < 0.005;

b
P < 0.025 (Student’s t-test, two-tailed).
J. A. MacDonald and K. B. Storey Temperature effects on hibernator PFK allostery
FEBS Journal 272 (2005) 120–128 ª 2004 FEBS 121
concentrations higher than 10 mm, phosphate pro-
duced inhibitory effects. A further increase in pH value
to 7.5 removed all activating characteristics of phos-
phate and it acted as an inhibitor with an I
50
value of
8.47 ± 0.21 mm. At pH 8.0, the inhibition was even
stronger with a decrease in the I
50
to 3.96 ± 0.36 mm.
Table 2 shows that PFK exhibited significantly dif-
ferent affinity for its F6P substrate at both high and
low temperatures. The enzyme showed sigmoidal F6P
kinetics at both temperatures with Hill coefficients of
 2 in the absence of added phosphate. However, the
S
0.5
for F6P was significantly lower (by 39%) at 5 °C
than at 37 °C (at 0 mm phosphate). PFK exhibits sig-
moidal F6P kinetics at lower pH values but converts
to hyperbolic kinetics with the addition of allosteric
activators (F1,6P
2
, F2,6P
2
, AMP, inorganic phosphate,

or NH
4
+
) or a rise in pH value to near 8 [17–19]. As
would be predicted for an activator, the addition of
10 mm phosphate to S. lateralis PFK in 37 °C assays
increased the maximal velocity by 1.5-fold, reduced the
S
0.5
by 27% and reduced the Hill coefficient to 1.21;
this low n
H
value indicates a hyperbolic relationship
between velocity and [F6P] (Table 2). However, the
effects of inorganic phosphate on F6P kinetics at 5 °C
and pH 7.5 were different. Figure 2 shows that the
F6P saturation curve was shifted strongly to the left
with the addition of as little as 5 mm inorganic phos-
phate and S
0.5
was reduced by 65% (Table 2). The
addition of phosphate also changed the V–[F6P] rela-
tionship from sigmoidal to hyperbolic; the Hill coeffi-
cient dropping by 50% in the presence of 5 mm
phosphate. However, unlike with the situation at
37 °C, increasing phosphate concentrations caused a
strong reduction in enzyme maximal velocity; V
max
was reduced by 29% at 5 mm and by 64% at 20 mm
phosphate (an effect that can also be seen in Fig. 1).

Effects of temperature and phosphate concentration
on the ATP kinetics of S. lateralis PFK are shown in
Table 3. The enzyme showed hyperbolic substrate sat-
uration kinetics with respect to Mg.ATP concentration
at both 5 and 37 °C with a much lower K
m
for
Mg.ATP at low temperature. The value at 5 °C was
only 15% of the value at 37 °C; hence, affinity for
both substrates, F6P and ATP, increased at low tem-
perature. As is common for PFK, ATP had inhibitory
effects at higher levels and inhibition was stronger at
37 °C with an I
50
for Mg.ATP of 1.29 mm compared
with 2.13 at 5 °C. The addition of phosphate at 37 °C
produced the typical effects of an activator on ATP
kinetics; 10 mm phosphate lowered the K
m
for
Mg.ATP by 58% and increased the I
50
by 2.2-fold.
Phosphate effects on ATP kinetics at 5 °C were differ-
ent. Phosphate had virtually no affect on the K
m
of
Fig. 1. Effects of pH and inorganic phosphate concentration on the
activity of S. lateralis phosphofructokinase at 5 °C. Activities are
expressed relative to the PFK activity in the absence of phosphate

at each pH value. PFK activity was measured as described in Mate-
rials and methods at pH 7.0 (j), pH 7.3 (r), pH 7.6 (d), and pH 8.0
(m). Data are means ±
SEM for n ¼ 3 separate determinations.
Table 2. The effect of inorganic phosphate concentration on fructose 6-phosphate kinetics of S. lateralis muscle PFK at two assay tempera-
tures. Maximal velocity values are milliunits of enzyme activity obtained for 0.2 lg (at 5 °C) or 0.03 lg (at 37 °C) of purified S. lateralis skel-
etal muscle phosphofructokinase. The pH values of the assay mixtures were fixed at 23 °C using 50 m
M imidazole–HCl buffer and were
then allowed to vary with temperature so that the pH at 5 °C was 7.5 and the pH at 37 °C was 7.2. The concentration of Mg.ATP was held
at 0.5 m
M. All other assay conditions are detailed in the Materials and methods. Data are means ± SEM, n ¼ 4 separate determinations.
Temperature Phosphate (m
M) S
0.5
F6P (mM) Hill coefficient (n
H
) Maximal velocity (mU)
5 °C 0 0.071 ± 0.009 2.30 ± 0.43 9.4 ± 0.3
5 0.025 ± 0.002
a
1.23 ± 0.13
b
6.7 ± 0.2
a
15 0.032 ± 0.004
a
1.12 ± 0.11
b
3.7 ± 0.1
a

20 0.022 ± 0.002
a
0.82 ± 0.15
b
3.4 ± 0.1
a
37 °C 0 0.121 ± 0.004 2.03 ± 0.21 6.21 ± 0.2
10 0.088 ± 0.007
b
1.21 ± 0.06
b
9.09 ± 0.4
a
a
Significantly different from the corresponding value at 0 mM phosphate using the Student’s t-test (two-tailed) or one-way analysis of
variance followed by the Student–Newman–Keuls test (two-tailed), P < 0.005,
b
P < 0.05.
Temperature effects on hibernator PFK allostery J. A. MacDonald and K. B. Storey
122 FEBS Journal 272 (2005) 120–128 ª 2004 FEBS
Mg.ATP at 5 °C, but it did alleviate ATP inhibition.
The I
50
for Mg.ATP increased by 2-fold in the pres-
ence of 5 mm phosphate and by 5-fold with 20 mm
phosphate. However, as was also seen in Table 2, the
maximum velocity of PFK at 5 °C was reduced in the
presence of phosphate by 49 and 79%, respectively, at
5 and 10 mm phosphate. This effect of phosphate on
PFK velocity is shown as a Hanes–Wolf plot in Fig. 3.

Phosphate inhibition was found to be noncompetitive
with respect to Mg.ATP and the K
i
was determined to
be 0.15 ± 0.01 mm (n ¼ 4).
Temperature effects on the activities of PFK from a
hibernating (ground squirrel) and nonhibernating (rab-
bit) mammal were investigated. Arrhenius plots were
constructed from V
max
values determined in assays run
in buffer only (50 mm imidazole) or in buffer plus
phosphate (50 mm imidazole plus 10 mm phosphate)
under optimal Mg.ATP conditions (0.5 mm) (Fig. 4).
The rabbit enzyme showed enhanced activity in the
presence of phosphate over the entire temperature
range tested (Fig. 4A). In contrast, the ground squirrel
enzyme was activated by phosphate at higher tempera-
tures, but at temperatures below 9 °C, phosphate
reduced enzyme velocity as compared with assays with-
out phosphate (Fig. 4B).
The temperature relationships of rabbit PFK in both
the presence and absence of phosphate exhibited sharp
breaks in the Arrhenius plot at 15 °C (Fig. 4A). The
activation energy (Ea) values for the 3–15 °C tempera-
ture range of the plots were 103.2 ± 0.2 and
80.0 ± 1.2 kJ mol
)1
in the absence and presence of
phosphate. For the 15–45 °C temperature range, the

Ea values were 25.7 ± 0.7 and 26.2 ± 1.1 kJ mol
)1
,
in the absence and presence of phosphate, respectively.
The temperature relationship for ground squirrel PFK
was linear from 3 °Cto28°C with an Ea value of
54.1 ± 0.8 kJ mol
)1
(n ¼ 4) when assayed in imida-
zole under optimal ATP concentrations (Fig. 4B).
When assayed in the presence of phosphate under opti-
mal Mg.ATP conditions, a very sharp break in the
Arrhenius relationship was seen at 12 °C; the Ea value
was 43.9 ± 0.2 kJ mol
)1
over the range from 12 to
45 °C and rose sharply by 2.8-fold to 122.2 ±
6.1 kJ mol
)1
(n ¼ 4) between 2 and 12 °C.
Temperature effects on ground squirrel PFK were
also assessed under inhibitory (but physiological) con-
centrations of ATP (Fig. 4C). In this situation, phos-
phate inhibition of ground squirrel PFK occurred at
all temperatures below 27 °C. The Ea for the linear
portion of the plot (3 °Cto29°C) was 61.9 ±
0.9 kJ mol
)1
(n ¼ 4). Temperature had little effect on
ground squirrel PFK activity under inhibitory

Mg.ATP concentrations when assayed in the absence
Fig. 2. The effect of inorganic phosphate on F6P kinetics of S. lat-
eralis PFK at 5 °C. PFK activity was measured as described in
Materials and methods with phosphate at: 0 m
M (j), 5 mM (r),
15 m
M (d), or 20 mM (m). Data shown are the result of one trial
but are representative of n ¼ 4 determinations from separate pre-
parations of enzyme.
Table 3. The effect of inorganic phosphate concentration on Mg.ATP kinetics of S. lateralis muscle PFK at two different temperatures. Maxi-
mal velocity values are milliunits of enzyme activity obtained for 0.2 lg (at 5 °C) or 0.03 lg (at 37 °C) of purified S. lateralis skeletal muscle
phosphofructokinase. The pH values of the assay mixtures were fixed at 23 °C using 50 m
M imidazole–HCl buffer and were then allowed to
vary with temperature so that the pH at 5 °C was 7.5 and the pH at 37 °C was 7.2. The concentration of F6P was held at 0.5 m
M. All other
assay conditions are detailed in the Materials and methods. Data are means ± S.E.M., n ¼ 4 separate determinations.
Temperature Phosphate (m
M)K
m
Mg.ATP (lM) I
50
Mg.ATP (mM) Maximal Velocity (mU)
5 °C 0 19.6 ± 0.37 2.13 ± 0.22 9.25 ± 0.37
5 17.7 ± 0.23
a
4.70 ± 0.50
a
4.72 ± 0.13
a
10 18.9 ± 0.37 10.7 ± 0.64

a,b
1.92 ± 0.31
a,b
37 °C 0 127.0 ± 6.6 1.29 ± 0.19 7.23 ± 0.51
10 53.7 ± 3.2
a
2.79 ± 0.09
a
7.04 ± 0.18
a
Significantly different from the corresponding value at 0 mM phosphate using one-way analysis of variance with the Student–Newman–
Keuls test (two-tailed), P < 0.01;
b
significantly different from the value with 5 mM phosphate.
J. A. MacDonald and K. B. Storey Temperature effects on hibernator PFK allostery
FEBS Journal 272 (2005) 120–128 ª 2004 FEBS 123
of phosphate; a slight decline in activity was observed
with increased temperature.
Figure 5 presents a plot of log W vs. 1 ⁄ T, where W
is the ratio of maximal velocity with 10 mm phosphate
to maximal velocity in the absence of phosphate [17].
Under optimal Mg.ATP levels, the data was character-
ized by a roughly linear relationship from 45 °Cto
9 °C with a slightly negative slope ()0.8). The tem-
perature at which phosphate had no allosteric effect
was 6.6 ± 0.04 °C(n ¼ 4). However, when the enzyme
was assayed in the presence of inhibitory levels of
Mg.ATP (5 mm), the relationship was characterized by
a sharp negative slope ()5.3) that crossed through zero
at 28.1 ± 0.1 °C(n ¼ 4).

Discussion
A reduction in glycolytic rate occurs during hiberna-
tion with the putative major site of inhibitory control
being phosphofructokinase. Major physiological mani-
festations during hibernation that are predicted from
the PFK control site include: (a) the suppression of
shivering thermogenesis; (b) a shift to a lipid-based
metabolism; (c) the conservation of muscle glycogen
stores for use during arousal, and (d) the stability of
plasma glucose levels [1,3,4]. In hibernators, the aci-
dotic conditions found in muscle during hibernation
can lead to an increase in histidine protonation and
these effects may be translated into alterations in
enzyme kinetics and structural properties which in turn
inhibit glycolysis [13,18,19]. Considerable work has
been completed in an effort to determine the mechan-
ism by which regulation of PFK occurs during
hibernation. PFK kinetic constants in S. lateralis skel-
etal muscle showed no differences when euthermic and
hibernating animals were compared [12]. This was in
contrast to results obtained from the small hibernators,
the meadow jumping mouse (Zapus hudsonius) [20],
and the little brown bat (Myotis lucifugus) (K. B.
Storey, unpublished data), which exhibited altered
PFK kinetic properties during hibernation that were
consistent with inactivation by post-translational modi-
fication. Another approach focused on temperature
and pH dependent shifts in PFK activity and subunit
assembly [6,7,21,22]. The effects of acidosis and tem-
perature were proposed to cause reversible inhibition

of PFK via inactive dimer formation. Previous work in
this laboratory [14] has questioned whether physiologi-
cally relevant pH changes under simulated in vivo con-
ditions of protein crowding affect PFK activity via
subunit assembly at hibernating temperatures. The
data presented here also conflict with the idea of tem-
perature and pH induced tetramer–dimer regulation; in
fact, the phosphate inhibition of PFK shown in Fig. 1
at low temperature decreased with decreasing pH.
Solute interactions have also been proposed to act
synergistically with the pH and temperature interactions
detailed above for the regulation of hibernator PFK.
Significant inactivation of PFK has been suggested to
occur at physiological levels of urea and inactivation
is proposed via unfolding of native macromolecules
through increased solvent exposure of subunit inter-
action sites [6,21]. However, the counteracting solute
theory proposed by Somero and coworkers has not been
shown to have major effects on the properties of PFK
either from estivating [23,24] or hibernating species [14].
Fig. 3. (A) The effect of inorganic phosphate on MgATP kinetics of S. lateralis PFK at 5 °C. PFK activity was measured as described in
Materials and methods with phosphate at: 0 m
M (j), 5 mM (m), and 10 mM (d). Data shown are the result of one trial but are representative
of n ¼ 4 determinations from separate preparations of enzyme. (B) Data replotted as a Hanes–Wolff plot showing the calculated K
i
for phos-
phate inhibition. Inset: Secondary plot of PFK maximal velocity vs. phosphate concentration at 5 °C. Data are means ±
SEM, n ¼ 4 separate
determinations.
Temperature effects on hibernator PFK allostery J. A. MacDonald and K. B. Storey

124 FEBS Journal 272 (2005) 120–128 ª 2004 FEBS
Allosteric activators and inhibitors are generally
considered to induce or bind to distinctly different
enzyme conformations and thereby convey altered
functionality to a regulatory enzyme. Allosteric modifi-
ers act either by changing the strength of subsequent
substrate binding by the enzyme or by changing the
activation energy of the enzyme-catalyzed reaction
[17]. The tissue-specific responses of PFK in S. lateralis
during hibernation illustrated the importance of allo-
steric activators in regulating PFK activity in heart
and leg muscle [12]. In the case of leg muscle, the con-
centration of the potent activator, F2,6P
2
, decreased
from a level five times the K
a
value in euthermic ani-
mals to one-half the K
a
value in hibernating animals,
suggesting that F2,6P
2
levels may influence glycolytic
rates by directly regulating PFK activity in these tis-
sues. Temperature effects on inorganic phosphate allo-
stery of PFK may also have a significant contribution
to the regulation of glycolytic metabolism in the skel-
etal muscle of this hibernating mammal.
Fig. 5. Plot of log W vs. 1 ⁄ T summarizing the effect of 10 mM

phosphate on V
max
for S. lateralis PFK under optimal (h) and inhibi-
tory (j) Mg.ATP levels. W is the ratio of maximal PFK velocity with
10 m
M phosphate to maximal PFK velocity in the absence of phos-
phate. Data are means ±
SEM, n ¼ 4 separate determinations.
Fig. 4. Arrhenius plots of skeletal muscle PFK activity vs. tempera-
ture under optimal or inhibitory ATP concentrations. (A) Rabbit PFK
measured under optimal substrate conditions: 1.0 m
M F6P, 0.5 mM
Mg.ATP, 5 mM MgCl
2
,50mM KCl, and 0.15 mM NADH with buffer
(d)50m
M imidazole, pH 7.5 at 5 °Cor(s)50mM imidazole
+10 m
M K
2
HPO
4
⁄ KH
2
PO
4
, pH 7.5 at 5 °C. (B) ground squirrel PFK
measured under the same conditions, and (C), ground squirrel
PFK measured under conditions of inhibitory Mg.ATP: 5.0 m
M

F6P, 5.0 mM Mg.ATP, 5 mM MgCl
2
,50mM KCl, and 0.15 mM
NADH with buffer (d)50mM imidazole, pH 7.5 at 5 °Cor(s)
50 m
M imidazole +10 mM K
2
HPO
4
⁄ KH
2
PO
4
, pH 7.5 at 5 °C. Data
are means ±
SEM, n ¼ 4 separate determinations.
J. A. MacDonald and K. B. Storey Temperature effects on hibernator PFK allostery
FEBS Journal 272 (2005) 120–128 ª 2004 FEBS 125
Kinetic findings interpreted for mammalian muscle
PFK have indicated the presence of not less than
seven substrate, inhibitor and de-inhibitor sites on the
enzyme [5]. ATP inhibition of PFK activity is over-
come by small increases in ADP, AMP and inorganic
phosphate, all of which increase in the cell whenever
ATP use exceeds ATP production. This effect is parti-
ally expressed in the low temperature F6P kinetics of
hibernator PFK; increasing phosphate levels initially
result in a hyperbolic shift in the F6P substrate curve
but with an unusual decrease in V
max

. However, fur-
ther phosphate addition does not have an effect on
substrate affinity and continues to lower V
max
. A sim-
ilar phenomenon was seen with respect to the allosteric
influences of phosphate on PFK Mg.ATP kinetics. The
effect of phosphate is pH dependent as lowering the
pH changes inhibition to activation (Fig. 1). It would
appear that phosphate inhibition is induced by low
temperature as the addition of phosphate at 37 °C
showed strong activation at a pH that showed inhibi-
tion at 5 °C. Experimental pH values determined
in hibernating skeletal muscle by in vivo
31
P NMR
spectroscopy [25] suggest that PFK inhibition could
occur under in vivo conditions.
The temperature-induced inversion of allosteric
phosphate effects is observed only at saturating
Mg.ATP concentrations. Unlike temperature-induced
changes in Mg.ADP allostery effects seen for F6P
kinetics for Bacillus stearothermophilus PFK [17], the
effects on hibernator PFK are due to a change in the
activation energy of the enzyme-catalyzed reaction
induced by the allosteric ligand and not by changes in
the extent to which the binding of allosteric ligand
modifies the affinity of enzyme for substrate. The
Hanes–Wolf plot in Fig. 3(B) conclusively demon-
strates that the K

m
value for Mg.ATP was not affected
by increasing phosphate levels at low temperature as
the data set was typical of noncompetitive inhibition.
Braxton et al. [17] previously defined the effect of an
allosteric ligand on V
max
via the use of Arrhenius plots
that graph the ratio of maximal velocities when the
allosteric ligand is saturating and when the allosteric
ligand is absent. The plot of W vs. 1 ⁄ T for S. lateralis
PFK under optimal conditions is relatively horizontal
and only crosses below 0 at temperatures less than
7 °C. So, under optimal conditions, the allosteric effect
of phosphate is consistent throughout the temperature
range investigated and has little effect on V
max
. How-
ever, under inhibitory concentrations of Mg.ATP, the
plot of log W vs. 1 ⁄ T is a linear relationship with a
sharply negative slope such that log W is equal to zero
at 29 °C (Fig. 5). This result demonstrates that tem-
perature has pronounced effects on phosphate allostery
of PFK with activating effects becoming inhibitory at
temperatures less than 29 °C. A comparison of the two
relationships indicate that the decrease in maximal
activity associated with phosphate is independent of
Q
10
effects. Interestingly, PFK from a nonhibernating

mammal (i.e. rabbit) lacks the temperature influences
on phosphate allostery (Fig. 4A).
It should be noted that the inhibitory levels of ATP
used are actually within the range of physiological
ATP concentrations present in ground squirrel skeletal
muscle during hibernation. ATP and other adenylates
have been quantified in S. lateralis after a week long
hibernation bout; the total adenylate pool decreased
in concentration without a corresponding decrease in
energy charge. ATP levels dropped by 29% to a hiber-
nating value of 2.9 mm [26] so that during hibernation,
at least over the short-term, [ATP] appears to remain
inhibitory with respect to temperature-induced phos-
phate inhibition. Thus, the response of PFK under
inhibitory ATP in the presence of added phosphate is
perhaps the closest mimic to the in vivo state.
Although a 0.3 unit pH difference exists between the
5 °C and 37 °C assay environments, we do not feel
that this alone explains the different kinetics observed.
It is well known that as PFK is subjected to higher
pH values, the S
0.5
for F6P decreases and the enzyme
looses allosteric properties [5]; however, our results
show that the sigmoidal character of the F6P kinetics
is retained at 5 °C at pH 7.5 and indicate that changes
in kinetic parameters were most probably due to tem-
perature alone. Sensitivity to adenylates (ATP inhibi-
tion, AMP and ADP activation) was reduced when the
enzyme was assayed at 5 °C, a temperature character-

istic of hibernation, as was sensitivity to F2,6P
2
. PFK
is generally thought of as being sensitive to two mes-
sages: (a) overall cellular energy status, via adenylate
and NH
4
+
levels, and (b) biosynthetic demands for
carbohydrates, via F2,6P
2
signaling that is the way
that extracellular hormones also influence PFK (via
reversible phosphorylation control over PFK-2, the
enzyme that synthesizes F2,6P
2
). Brooks and Storey
[12] observed that the concentration of F2,6P
2
in
hibernating golden-mantled ground squirrel leg muscle
decreased to 20% of the euthermic value. The 11-fold
increase in the K
a
value for F2,6P
2
at 5 °C when cou-
pled with this decrease in tissue F2,6P
2
levels would

effectively eliminate regulatory effects on PFK by this
allosteric molecule during hibernation. The data also
indicate that the enzyme is less likely to be activated
by rising AMP and ADP in the torpid muscle (hence,
muscle glycolysis is less sensitive to changing energetic
state when torpid) and is clearly much less sensitive
to biosynthetic signals (F2,6P
2
) which would help to
Temperature effects on hibernator PFK allostery J. A. MacDonald and K. B. Storey
126 FEBS Journal 272 (2005) 120–128 ª 2004 FEBS
preserve carbohydrate reserves during torpor. This
enzyme is particularly well suited to the metabolic
conditions of hibernation as a decreased response to
adenylates and to F2,6P
2
as well as the increased cit-
rate levels that accompany a switch to fatty acid oxida-
tion would all serve to suppress PFK activity and
permit carbohydrate sparing during hibernation.
In summary, we suggest that temperature-induced
alterations in PFK activity via phosphate allostery
could be a means by which suppression of PFK activ-
ity, and hence glycolytic flux, occurs during metabolic
depression in mammalian hibernators. Our results
demonstrate that PFK is less likely to be activated in
cold torpid muscle, PFK is less sensitive to changing
adenylate levels at low temperatures characteristic of
torpor, and PFK is clearly much less sensitive to bio-
synthetic signals. All of these characteristics of hiber-

nator PFK would serve to reduce glycolytic rate and
help to preserve carbohydrate reserves during torpor.
Materials and methods
Animals and chemicals
Adult golden-mantled ground squirrels, S. lateralis, were
obtained from the Crooked Creek area of the White Moun-
tains of California. Details of animal holding, feeding and
hibernation were described in [15]. All possible measures
were taken to minimise pain and discomfort during animal
euthanasia in accordance with protocols approved by the
Carleton University Animal Care and Use Committee.
Squirrels were killed by decapitation and hind leg skeletal
muscle was quickly excised and flash frozen in liquid nitro-
gen. Tissues were transported to Carleton University on
dry ice and were then stored at )80 °C until use. All bio-
chemicals and coupling enzymes were obtained from Boeh-
ringer Mannheim (Montreal, PQ) or Sigma Chemical Co.
Purification and standard assay of
phosphofructokinase
PFK (EC 2.7.1.11) was purified from skeletal muscle of
euthermic ground squirrels as described previously [14].
Rabbit skeletal muscle PFK was purified following a proce-
dure modified from Ramadoss et al. [27]. Purified PFK was
used immediately or stored for up to a week in 30% (v ⁄ v)
glycerol at )20 °C. PFK activity was measured by a cou-
pled enzyme assay [14] and the change in absorbance at
340 nm as a result of NADH consumption was monitored
with a Dynatech MR5000 microplate reader with biolynx
data capture software. Enzyme activities were analyzed with
a microplate analysis program [28] and kinetic param-

eters were determined using a simple computer program
[29]. Assay temperature was manipulated by using the
microplate thermal controller for 37 °C assays or by placing
the entire microplate reader into a low temperature incuba-
tor for 5 °C studies; in the latter case, thermistors placed in
selected wells and attached to a YSI Model 42 SL telether-
mometer were used to confirm assay temperature. All reac-
tions were initiated with the addition of purified PFK.
Standard assay conditions were: 20 mm imidazole-HCl
buffer, 5 mm MgCl
2
,50mm KCl, 0.2 mm F6P, 0.5 mm
Mg.ATP, 10 mm 2-mercaptoethanol, 0.15 mm NADH, and
1 U each of aldolase, triosephosphate isomerase and gly-
cerol-3-phosphate dehydrogenase. Ammonium sulfate was
removed from the coupling enzymes by centrifugation
through a small (5 mL) column of Sephadex G-25 equili-
brated in 20 mm imidazole-HCl buffer pH 7.2 (at 37 °C)
containing 5 mm MgCl
2
and 10 mm 2-mercaptoethanol
[30]. Imidazole buffer pH was adjusted at 23 °C to produce
pH values of 7.5 or 7.2 at 5 °Cor37°C, respectively; this
was calculated assuming a +0.017 unit increase in pH per
1 °C decrease for imidazole buffer [14]. Due to the sensitiv-
ity of PFK to minor pH variation, the pH of the inorganic
phosphate solutions were also adjusted to be pH 7.5 at
5 °C or pH 7.2 at 37 °C.
Protein concentration was determined by the Coomassie
blue G-250 dye binding method using the Bio-Rad pre-

pared reagent and bovine serum albumin as the standard
[31].
Acknowledgements
The authors thank Dr Craig Frank, Fordham Univer-
sity for supplying the ground squirrel tissues and J. M.
Storey for critical commentary on the manuscript. The
work was supported by an N.S.E.R.C. Canada discov-
ery grant (KBS) and postgraduate scholarship (JAM).
JAM is currently the holder of a Protein Engineering
Network of Centres of Excellence Chair in Protein
Sciences.
References
1 Wang LCH & Lee TF (1996) Torpor and hibernation in
mammals: metabolic, physiological and biochemical
adaptations. In Handbook of Physiology: Environmen-
tal Physiology. (Fregley MJ & Blatteis CM, eds), Sec. 4,
Vol. 1, pp. 507–532. Oxford University Press, New
York, NY.
2 Hochachka PW & Somero GN (1984) Biochemical
Adaptation. Princeton University Press, Princeton, NJ.
3 Carey HV, Andrews MT & Martin SL (2003) Mamma-
lian hibernation: cellular and molecular responses to
depressed metabolism and low temperature. Physiol Rev
83, 1153–1181.
4 Wang, LCH (1989) Ecological, physiological and bio-
chemical aspects of torpor in mammals and birds. In
J. A. MacDonald and K. B. Storey Temperature effects on hibernator PFK allostery
FEBS Journal 272 (2005) 120–128 ª 2004 FEBS 127
Comparative and Environmental Physiology. 4: Animal
Adaptation to Cold (Wang LCH, ed.), pp. 361–401.

Springer-Verlag, Berlin, Germany.
5 Kemp RG & Foe LG (1983) Allosteric regulatory prop-
erties of muscle phosphofructokinase. Mol Cell Biochem
57, 147–154.
6 Hand SC & Somero GN (1983) Phosphofructokinase of
the hibernator Citellus beecheyi, temperature and pH
regulation of activity via influences on the tetramer-
dimer equilibrium. Physiol Zool 56, 280–388.
7 Bock PE & Frieden C (1976) Phosphofructokinase:
mechanism of the pH-dependent inactivation and reacti-
vation of the rabbit muscle enzyme. J Biol Chem 251,
5630–5636.
8 Bock PE & Frieden C (1976) Phosphofructokinase: role
of ligands in pH-dependent structural changes of the
rabbit muscle enzyme. J Biol Chem 251, 5637–5643.
9 Benoit MA, Debauche P & Devos PK (1994) Phospho-
fructokinase from the posterior gills of the euryhaline
crab, Eriocheir sinensis: evidence for its regulation by
phosphorylation. J Comp Physiol B 164, 165–171.
10 Brooks SPJ & Storey KB (1991) A quantitative evalua-
tion of the effect of enzyme complexes on the glycolytic
rate in vivo: mathematical modeling of the glycolytic
complex. J Theor Biol 149 , 361–375.
11 Brooks SPJ & Storey KB (1988) Re-evaluation of the
‘glycolytic complex’ in muscle: a multi-technique
approach using trout white muscle. Arch Biochem
Biophys 267, 13–22.
12 Brooks SPJ & Storey KB (1992) Mechanisms of glyco-
lytic control during hibernation in the ground squirrel
Spermophilus lateralis. J Comp Physiol B 162, 23–28.

13 Malan A, Rodeau JL & Daull F (1985) Intracellular pH
in hibernation and respiratory acidosis in the European
hampster. J Comp Physiol B 156, 251–258.
14 MacDonald JA & Storey KB (2001) Reassessment of
the cold labile nature of phosphofructokinase from a
hibernating ground squirrel. Mol Cell Biochem 225,
51–57.
15 MacDonald JA & Storey KB (2002) Purification and
characterization of fructose bisphosphate aldolase from
the ground squirrel, Spermophilus lateralis: Enzyme role
in mammalian hibernation. Arch Biochem Biophys 408,
279–285.
16 Storey KB & Storey JM (2000) Gene expression and
protein adaptations in mammalian hibernation. In Life
in the Cold (Heldmaier G & Klingenspor M, eds),
pp. 303–313. Springer-Verlag, Heidelberg, Germany.
17 Braxton BL, Tlapak-Simmons VL & Reinhart GD
(1994) Temperature induced inversion of allosteric
phenomena. J Biol Chem 269, 47–50.
18 Malan, A. (1982) Respiration and acid-base state in
hibernation. In Hibernation and Torpor in Mammals
and Birds (Lyman CP, Willis JS, Malan A & Wang LCH,
eds), pp. 273–282. Academic Press, New York, NY.
19 Malan A (1986) pH as a central factor in hibernation.
In Living in the Cold: Physiological and Biochemical
Adaptations (Heller HC, Musacchia XJ & Wang LCH,
eds), pp. 61–70. Elsevier, New York, NY.
20 Storey KB (1987) Regulation of liver metabolism by
enzyme phosphorylation during mammaliam hiberna-
tion. J Biol Chem 262, 1670–1673.

21 Hand SC & Somero GN (1982) Urea and methylamine
effects on rabbit muscle phosphofructokinase. Catalytic
stability and aggregation state as a function of pH and
temperature. J Biol Chem 257, 734–741.
22 Somero GN, Lowery MS & Roberts SJ (1991) Com-
partmentalization of animal enzymes: physiological and
evolutionary significance. Am Zool 31, 493–503.
23 Grundy JE & Storey KB (1994) Urea and salt effects on
enzymes from estivating and non-estivating amphibians.
Mol Cell Biochem 131, 9–17.
24 Cowan KJ & Storey KB (2002) Urea and KCl have dif-
ferential effects on enzyme activities in liver and muscle
of estivating versus non-estivating species. Biochem Cell
Biol 80, 745–755.
25 McArthur MD, Hanstock CC, Malan A, Wang LCH &
Allen PS (1990) Skeletal muscle pH dynamics during
arousal from hiberantion measured by
31
P NMR spec-
troscopy. J Comp Physiol B 160, 339–347.
26 MacDonald JA & Storey KB (1999) Regulation of
ground squirrel Na
+
K
+
-ATPase activity by reversible
phosphorylation during hibernation. Biochem Biophys
Res Commun 254 , 424–429.
27 Ramadoss CS, Luby LJ & Uyeda K (1976) Affinity
chromatography of phosphofructokinase. Arch Biochem

Biophys. 175, 487–494.
28 Brooks SPJ (1994) A program for analyzing the enzyme
rate data obtained from a microplate reader. Biotech-
niques 17, 1154–1161.
29 Brooks SPJ (1992) A simple computer program with
statistical tests for the analysis of enzyme kinetics. Bio-
techniques 13, 906–911.
30 Helmerhorst E & Stokes HH (1980) Microcentrifuge
desalting: a rapid, quantitative method for desalting
small amounts of protein. Anal Biochem 104, 130–135.
31 Bradford MM (1976) A rapid, sensitive method for the
quantification of microgram quantities of protein util-
izing the principle of protein-dye binding. Anal Biochem
72, 248–254.
Temperature effects on hibernator PFK allostery J. A. MacDonald and K. B. Storey
128 FEBS Journal 272 (2005) 120–128 ª 2004 FEBS