Storey, Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6 Canada E-mail: kenneth_storey@carleton.ca Received 7 July 200
Trang 1phenomena of phosphofructokinase from a hibernating ground squirrel (Spermophilus lateralis)
Justin A MacDonald1and Kenneth B Storey2
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 37C 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 0–5C if ambient temperature
falls below 0C) 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: kenneth_storey@carleton.ca
(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 37C and 5 C, values character-istic of body temperatures in euthermia vs hibernation The enzyme showed reduced sensitivity to all activators at 5C, the Ka values for AMP, ADP, NH4+ and F2,6P2 were 3–11-fold higher at 5C than at
37C 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 5C Decreasing temperature alone causes the actions of inorganic phosphate to change from activation to inhibition We found that Kmvalues for ATP remained constant while Vmax dropped sig-nificantly upon the addition of phosphate Phosphate inhibition at 5C was noncompetitive with respect to ATP and the Kiwas 0.15 ± 0.01 mm (n¼ 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.
Trang 2[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 gluchex-ose) 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 hibernainhibi-tion 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 30C 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-eralismuscle were compared at assay temperatures and
pH values that mimic the hibernating (5C, pH 7.5)
vs euthermic (37C, pH 7.2) conditions found in vivo
in S lateralis skeletal muscle Table 1 shows the effect
of temperature on activation coefficient (Ka) values for allosteric activators and the values of concentration of the inhibitor that reduces control activity by 50% (I50) The enzyme showed reduced sensitivity to all activa-tors at 5C, the Ka values for AMP, ADP, NH4+, and F2,6P2 being 5.3-, 3.4-, 2.6- and 11.3-fold higher
at 5C, respectively, compared with the corresponding
37C 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; I50values dropped to 53 and 60%, respectively, of the corresponding values at
37C
Inorganic phosphate is typically an activator of PFK, and Table 1 shows that the ground squirrel enzyme responded to phosphate as expected at 37C with a Ka value of 2.0 ± 0.2 mm However, inorganic phosphate was unable to elicit an activation of PFK activity at 5C; in fact, the addition of inorganic phos-phate resulted in an inhibition of PFK activity Fur-ther analysis of phosphate effects on PFK at 5C 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 Ka for phosphate was 1.73 ± 0.13 mm (n¼ 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 Ka and I50 determinations For I50 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.
KaFructose-2,6-bisphosphate (n M ) 39.9 ± 2.9 449 ± 15 a
K a Inorganic phosphate (m M ) 2.0 ± 0.2 NA
I50Phosphoenolpyruvate (m M ) 1.2 ± 0.1 0.64 ± 0.04 a
I 50 Mg.citrate (l M ) 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).
Trang 3concentrations 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 I50value of
8.47 ± 0.21 mm At pH 8.0, the inhibition was even
stronger with a decrease in the I50to 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
S0.5 for F6P was significantly lower (by 39%) at 5C
than at 37C (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 (F1,6P2, F2,6P2, AMP, inorganic phosphate,
or NH4+) 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 37C assays increased the maximal velocity by 1.5-fold, reduced the
S0.5 by 27% and reduced the Hill coefficient to 1.21; this low nH value indicates a hyperbolic relationship between velocity and [F6P] (Table 2) However, the effects of inorganic phosphate on F6P kinetics at 5C 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 S0.5 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
37C, increasing phosphate concentrations caused a strong reduction in enzyme maximal velocity; Vmax 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 Km for Mg.ATP at low temperature The value at 5C was only 15% of the value at 37C; 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
37C with an I50 for Mg.ATP of 1.29 mm compared with 2.13 at 5C The addition of phosphate at 37 C produced the typical effects of an activator on ATP kinetics; 10 mm phosphate lowered the Km for Mg.ATP by 58% and increased the I50 by 2.2-fold Phosphate effects on ATP kinetics at 5C were differ-ent Phosphate had virtually no affect on the Km 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 ) S0.5F6P (m M ) Hill coefficient (nH) Maximal velocity (mU)
a Significantly different from the corresponding value at 0 m M 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.
Trang 4Mg.ATP at 5C, but it did alleviate ATP inhibition.
The I50 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 5C 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 Ki was determined to
be 0.15 ± 0.01 mm (n¼ 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 Vmaxvalues 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 9C, 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 15C (Fig 4A) The activation energy (Ea) values for the 3–15C 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 15–45C 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 3C to 28 C with an Ea value of 54.1 ± 0.8 kJ mol)1 (n¼ 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 12C; the Ea value was 43.9 ± 0.2 kJ mol)1 over the range from 12 to
45C and rose sharply by 2.8-fold to 122.2 ± 6.1 kJ mol)1(n¼ 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 27C The Ea for the linear portion of the plot (3C to 29 C) was 61.9 ± 0.9 kJ mol)1 (n¼ 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 m M (r),
15 m M (d), or 20 m M (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.
a Significantly different from the corresponding value at 0 m M phosphate using one-way analysis of variance with the Student–Newman– Keuls test (two-tailed), P < 0.01;bsignificantly different from the value with 5 m M phosphate.
Trang 5of 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 45C to
9C with a slightly negative slope ()0.8) The
tem-perature at which phosphate had no allosteric effect
was 6.6 ± 0.04C (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 ± 0.1C (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 m M (m), and 10 m M (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 phosphos-phate concentration at 5 C Data are means ± SEM , n ¼ 4 separate determinations.
Trang 6Allosteric 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, F2,6P2, decreased from a level five times the Ka value in euthermic ani-mals to one-half the Ka value in hibernating animals, suggesting that F2,6P2 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 m M
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 m M
Mg.ATP, 5 m M MgCl2, 50 m M KCl, and 0.15 m M NADH with buffer (d) 50 m M imidazole, pH 7.5 at 5 C or (s) 50 m M 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 m M Mg.ATP, 5 m M MgCl 2 , 50 m M KCl, and 0.15 m M
NADH with buffer (d) 50 m M imidazole, pH 7.5 at 5 C or (s)
50 m M imidazole +10 m M K2HPO4⁄ KH 2 PO4, pH 7.5 at 5 C Data are means ± SEM , n ¼ 4 separate determinations.
Trang 7Kinetic 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 Vmax However,
fur-ther phosphate addition does not have an effect on
substrate affinity and continues to lower Vmax 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 37C
showed strong activation at a pH that showed
inhibi-tion at 5C Experimental pH values determined
in hibernating skeletal muscle by in vivo 31P 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 Kmvalue 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 Vmax 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
7C So, under optimal conditions, the allosteric effect
of phosphate is consistent throughout the temperature
range investigated and has little effect on Vmax
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 29C (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 29C A comparison of the two relationships indicate that the decrease in maximal activity associated with phosphate is independent of
Q10 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
5C 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 S0.5 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 5C 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 5C, a temperature character-istic of hibernation, as was sensitivity to F2,6P2 PFK
is generally thought of as being sensitive to two mes-sages: (a) overall cellular energy status, via adenylate and NH4+ levels, and (b) biosynthetic demands for carbohydrates, via F2,6P2 signaling that is the way that extracellular hormones also influence PFK (via reversible phosphorylation control over PFK-2, the enzyme that synthesizes F2,6P2) Brooks and Storey [12] observed that the concentration of F2,6P2 in hibernating golden-mantled ground squirrel leg muscle decreased to 20% of the euthermic value The 11-fold increase in the Kavalue for F2,6P2 at 5C when cou-pled with this decrease in tissue F2,6P2 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 (F2,6P2) which would help to
Trang 8preserve carbohydrate reserves during torpor This
enzyme is particularly well suited to the metabolic
conditions of hibernation as a decreased response to
adenylates and to F2,6P2 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 37C assays or by placing the entire microplate reader into a low temperature incuba-tor for 5C 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 MgCl2, 50 mm 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 37C) containing 5 mm MgCl2 and 10 mm 2-mercaptoethanol [30] Imidazole buffer pH was adjusted at 23C to produce
pH values of 7.5 or 7.2 at 5C or 37 C, respectively; this was calculated assuming a +0.017 unit increase in pH per
1C 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
5C 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
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