Muscle-type 6-phosphofructo-1-kinase and aldolase associate conferring catalytic advantages for both enzymes.
ABSTRACT 6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism.
- SourceAvailable from: Bhavapriya Vaitheesvaran[Show abstract] [Hide abstract]
ABSTRACT: Previous studies have demonstrated that glucose disposal is increased in the Fyn knockout (FynKO) mice due to increased insulin sensitivity. FynKO mice also display fasting hypoglycaemia despite decreased insulin levels, which suggested that hepatic glucose production was unable to compensate for the increased basal glucose utilization. The present study investigates the basis for the reduction in plasma glucose levels and the reduced ability for the liver to produce glucose in response to gluconeogenic substrates. FynKO mice had a 5-fold reduction in phosphoenolpyruvate carboxykinase (PEPCK) gene and protein expression and a marked reduction in pyruvate, pyruvate/lactate-stimulated glucose output. Remarkably, de novo glucose production was also blunted using gluconeogenic substrates that bypass the PEPCK step. Impaired conversion of glycerol to glucose was observed in both glycerol tolerance test and determination of the conversion of (13)C-glycerol to glucose in the fasted state. α-glycerol phosphate levels were reduced but glycerol kinase protein expression levels were not changed. Fructose-driven glucose production was also diminished without alteration of fructokinase expression levels. The normal levels of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate observed in the FynKO liver extracts suggested normal triose kinase function. Fructose-bisphosphate aldolase (aldolase) mRNA or protein levels were normal in the Fyn-deficient livers, however, there was a large reduction in liver fructose-6-phosphate (30-fold) and fructose-1,6-bisphosphate (7-fold) levels as well as a reduction in glucose-6-phosphate (2-fold) levels. These data suggest a mechanistic defect in the allosteric regulation of aldolase activity.PLoS ONE 01/2013; 8(11):e81866. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Cells are capable of metabolizing a variety of carbon substrates, including glucose, fatty acids, ketone bodies, and amino acids. Cellular fuel choice not only fulfills specific biosynthetic needs, but also enables programmatic adaptations to stress conditions beyond compensating for changes in nutrient availability. Emerging evidence indicates that specific switches from utilization of one substrate to another can have protective or permissive roles in disease pathogenesis. Understanding the molecular determinants of cellular fuel preference may provide insights into the homeostatic control of stress responses, and unveil therapeutic targets. Here, we highlight overarching themes encompassing cellular fuel choice; its link to cell fate and function; its advantages in stress protection; and its contribution to metabolic dependencies and maladaptations in pathological conditions.Trends in cell biology 09/2013; · 12.12 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Diabetes mellitus is characterized by hyperglycemia and its associated complications, including cardiomyopathy. Metformin, in addition to lowering blood glucose levels, provides cardioprotection for diabetic subjects. Glycolysis is essential to cardiac metabolism and its reduction may contribute to diabetic cardiomyopathy. Hexokinase (HK) and phosphofructokinase (PFK), rate-limiting enzymes of glycolysis, are downregulated in cardiac muscle from diabetic subjects, playing a central role on the decreased glucose utilization in the heart of diabetic subjects. Thus, the aim of this study was to determine whether metformin modulates heart HK and PFK from diabetic mice. Diabetes was induced by streptozotocin injection on male Swiss mice, which were treated for three consecutive days with 250 mg/kg metformin before evaluating HK and PFK activity, expression, and intracellular distribution on the heart of these subjects. We show that metformin abrogates the downregulation of HK and PFK in the heart of streptozotocin-induced diabetic mice. This effect is not correlated to alteration on the enzymes' transcription and expression. However, the intracellular distribution of both enzymes is altered in diabetic hearts that show increased activity of the soluble fraction when compared to the particulate fraction. Moreover, this pattern is reversed upon the treatment with metformin, which is correlated with the effects of the drug on the enzymes activity. Altogether, our results support evidences that metformin alter the intracellular localization of HK and PFK augmenting glucose utilization by diabetic hearts and, thus, conferring cardiac protection to diabetic subjects.International Union of Biochemistry and Molecular Biology Life 06/2012; 64(9):766-74. · 2.79 Impact Factor
Muscle-Type 6-Phosphofructo-1-kinase and Aldolase Associate
Conferring Catalytic Advantages for Both Enzymes
Mariah Celestino Marcondes, Mauro Sola-Penna, Renan da Silva Gianoti Torres, and Patricia Zancan
Laborato ´rio de Oncobiologia Molecular (LabOMol) and Laborato ´rio de Enzimologia e Controle do Metabolismo
(LabECoM), Departamento de Fa ´rmacos, Faculdade de Farma ´cia, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brazil
6-Phosphofructo-1-kinase (PFK) and aldolase are two se-
quential glycolytic enzymes that associate forming heterote-
tramers containing a dimer of each enzyme. Although free PFK
dimers present a negligible activity, once associated to aldolase
these dimers are as active as the fully active tetrameric confor-
mation of the enzyme. Here we show that aldolase-associated
PFK dimers are not inhibited by clotrimazole, an antifulgal az-
ole derivative proposed as an antineoplastic drug due to its in-
hibitory effects on PFK. In the presence of aldolase, PFK is not
modulated by its allosteric activators, ADP and fructose-2,6-bis-
phosphate, but is still inhibited by citrate and lactate. The asso-
ciation between the two enzymes also results on the twofold
stimulation of aldolase maximal velocity and affinity for its sub-
strate. These results suggest that the association between PFK
and aldolase confers catalytic advantage for both enzymes and
may contribute to the channeling of the glycolytic metabo-
? 2011 IUBMB
IUBMB Life, 63(6): 435–445, 2011
glycolysis; phosphofructokinase; aldolase; clotrimazole;
6-Phosphofructo-1-kinase (PFK; phosphofructokinase; EC
220.127.116.11) is the major regulatory glycolytic enzyme and acts as
the pacemaker of glycolysis (1). This highly regulated enzyme
exists in diverse oligomeric conformations, including mono-
mers, dimers, tetramers, and hexadecamers (1). The transition
between dimers and tetramers is highly relevant for the
enzyme’s regulation because the former have very low catalytic
activity, whereas the latter have been described as fully active
(1–3). Several allosteric modulators of PFK affect the equilib-
rium between dimers and tetramers; the inhibitors citrate and
lactate favor the formation of dimers, and the activators ADP
and fructose-2,6-bisphosphate (F2,6BP) stabilize tetramers (4–
8). Moreover, the association of PFK with aldolase or calmodu-
lin stabilizes PFK in a dimeric conformation that has a catalytic
activity equivalent to that of the tetramers (4, 9–11).
Aldolase (EC 18.104.22.168), the sequential enzyme to PFK on
glycolysis, cleaves the product of the PFK reaction, fructose-
and dihydroxyacetone phosphate. The association between PFK
and aldolase contributes to the channeling of glycolysis,
increasing the rate of this pathway (12–15). Aldolase and PFK
associate with the cytoskeleton (9, 16–23), especially in tumor
cells (19, 20), which increases their activity and the channeling
of glycolysis (14). This event is directly correlated to the War-
burg effect, conferring invasive metastatic properties to the tu-
mor (24, 25). The down-regulation of both PFK and aldolase
has been reported to drastically decrease tumor cell viability
Clotrimazole (CTZ) is an antifungal derivative azole with
calmodulin antagonist properties and described as a potential
antineoplasic drug due to its ability to decrease tumor cell gly-
colysis (20, 26–28). We have previously reported that this drug
decreases the association of PFK and aldolase with the tumor
cell cytoskeleton, thus decreasing tumor viability (20). More-
over, CTZ directly inhibits PFK by promoting the dissociation
of PFK tetramers into dimers (23) and by augmenting the inhib-
itory effectiveness of ATP (29).
The present work aimed to evaluate whether the interaction
of PFK and aldolase interferes with their catalytic activities and
the inhibition of the enzymes by CTZ.
Address correspondence to: Patricia Zancan, Laborato ´rio de Oncobio-
logia Molecular (LabOMol), Departamento de Fa ´rmacos, Faculdade de
Farma ´cia, Universidade Federal do Rio de Janeiro, Ilha do Funda ˜o, Rio
de Janeiro, RJ 21941-590, Brazil. Tel: 155-21-2260-9192, Ext. 203.
Received 17 February 2011; accepted 7 March 2011
ISSN 1521-6543 print/ISSN 1521-6551 online
IUBMBLife, 63(6): 435–445, June 2011
MATERIALS AND METHODS
ATP, ADP, CTZ, citrate, F1,6BP, F2,6BP, F6P, lactate,
NADH, triosephosphate isomerase, and a-glycerophosphate de-
hydrogenase were purchased from Sigma Chemical (St. Louis,
MO).32Pi was purchased from Instituto de Pesquisas Energe ´ti-
cas e Nucleares (Sa ˜o Paulo, Brazil). [c-32P]ATP was prepared
according to Maia et al. (30). PFK was purified from rabbit
skeletal muscle, as previously described (21). Aldolase from
rabbit skeletal muscle was purchased from Sigma Chemical (St.
Louis, MO). All protein content measurements were performed
as described by Lowry et al. (31).
PFK activity was measured by the method described by
Sola-Penna et al. (32) with the modifications introduced by Zan-
can and Sola-Penna (33, 34). The reaction medium contained
50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM [c-32P]ATP (4
lCi/nmol), 1 mM fructose-6-phosphate and 1 lg/mL PFK,
except when otherwise specified. The reaction was stopped after
increasing reaction times by the addition of a suspension of
activated charcoal in 0.1 M HCl and 0.5 M mannitol, and after
centrifugation, the supernatant containing [1-32P]fructose-1,6-
bisphosphate was analyzed in a liquid scintillation counter. The
signals from appropriate blanks in the absence of fructose-6-
phosphate were measured and subtracted from all measurements
to account for ATP hydrolysis. The catalytic rate was calculated
by linear regression of the amount of F1,6BP formed versus
reaction time. The enzyme activity was expressed as mU of
PFK activity, where 1 mU was taken to be the formation of 1
nmol of fructose-1,6-bisphosphate per minute of reaction.
Aldolase activity was assessed through a coupled enzyme
linked assay in a reaction medium containing 50 mM Tris-HCl
(pH 7.4), 5 mM MgCl2, 1 mM F1,6BP, 2 mM NADH, triose-
phosphate isomerase, and a-glycerophosphate dehydrogenase.
The reaction was followed spectrophotometrically at 340 nm,
which corresponds to the absorbance of NADH. Aldolase activ-
ity was expressed as mU, where 1 mU was considered to be the
reduction of 2 nmol of NADH per minute of reaction.
Intrinsic Fluorescence Measurements
Intrinsic fluorescence measurements of PFK were performed
described for the radioassay. Excitation wavelength was fixed at
280 nm, and fluorescence emission was scanned from 300 to
400 nm. The center of mass of the intrinsic fluorescence spectra
(CM) was calculated using:
where k is the wavelength and Ikis the fluorescence intensity at
a given k. Center of mass is used to evaluate the oligomeric
state of PFK because the dissociated enzyme exposes its trypto-
phans to the aqueous milieu to a greater extent than the
oligomer; thus, the fluorescence emitted by these tryptophans is
of lower energy. Consequently, the center of mass of a popula-
tion of tetramers is smaller than that of a population of dimers,
as confirmed in many recent publications (4–6, 11, 23, 29, 35).
Light Scattering Measurements
Light scattering measurements were performed as described
previously (6) using the same conditions described for the
radioassay. Appropriate reference spectra were subtracted from
the data to correct for background interferences. The excitation
wavelength was set at 510 nm and the emission light scattered
was scanned from 500 to 520 nm. The total concentration of
protein in the assay was 1 lg/mL.
Statistics and Calculations
Statistical analyses and enzyme kinetics calculations were
performed using the software SigmaPlot 10.0 integrated with
SigmaStat 3.51 (Systat, CA). Student’s t-tests or one-tailed
ANOVAs were used to evaluate the significance of the results.
P \ 0.05 was considered to be statistically significant.
The kinetic parameters for the inhibitory effects of CTZ on
PFK and aldolase were calculated as described previously (29)
by nonlinear regression using the equation
0:5þ ½CTZ?þ vR
where v is the calculated enzyme’s activity at a given concen-
tration of CTZ ([CTZ]), v0 is the difference between the
enzyme’s activity in the absence of CTZ and the activity when
CTZ is promoting its maximal inhibitory effects (vR), I0.5is the
inhibition constant equivalent to the concentration of CTZ
resulting in 50% of the maximal inhibition (vR) and n is the
Kinetic parameters for the effects of ATP on PFK were cal-
culated considering the two components for PFK modulation by
this metabolite. The first component is the stimulatory compo-
nent for the substrate saturation curve, in which PFK exhibits
an allosteric pattern that is described by the equation:
where v is the PFK activity at a given concentration of ATP
([ATP]), Vmaxappis the apparent maximal velocity calculated,
K0.5is the affinity constant for this component and nsis the
cooperativity index for this component. The second component
is the inhibitory component that can be adjusted by the equation:
436MARCONDES ET AL.
where v is the PFK activity at a given concentration of ATP
([ATP]), I0.5is the affinity constant for this component, niis the
cooperativity index for this component and Vsatis the PFK activ-
ity when the first component is saturated. Assuming this state-
ment, Vsatis a function of the first component of the curve and
can be substituted by Eq. (3) to result in the following equation:
which was fitted to the experimental data through nonlinear
regression for the effects of ATP on PFK activity.
Kinetic parameters for the effects of F6P on PFK were cal-
culated through nonlinear regression using the experimental
data to fit the parameters of the equation:
where v is the PFK activity calculated for a given concentration
of F6P ([F6P]), Vmaxis the maximal velocity calculated at satu-
rating concentrations of F6P, K0.5 is the affinity constant for
F6P, which is equal to the concentration of F6P responsible for
half-activation of the PFK by F6P, and n is the cooperativity
index for this phenomenon.
Kinetic parameters for aldolase were assessed using Eq. (6),
substituting F6P by F1,6BP.
The kinetic parameters for the stimulatory effects of PFK on
aldolase activity were calculated by nonlinear regression using
v ¼ viþ
where v is the calculated aldolase activity at a given concentra-
tion of PFK ([PFK]), viis the aldolase activity in the absence of
PFK, Ka is the activation constant reflecting the concentration
of PFK that results in 50% of the maximal activation (va) and n
is the cooperativity index.
Counteraction of CTZ-Induced Inhibition of PFK
PFK is inhibited by CTZ in a dose-dependent manner, with
an I0.5of 28 6 2 lM and a maximal inhibition of 70% (Fig.
1A, filled circles). However, when these experiments were per-
formed in the presence of 1 lg/mL aldolase, no inhibition of
PFK activity was observed up to 200 lM CTZ (Fig. 1A, empty
circles). We have reported that PFK inhibition by CTZ is due
to the ability of the drug to promote the dissociation of the fully
active tetrameric conformation of the enzyme into relatively
inactive dimers (23, 29). It has been reported that aldolase binds
to PFK, stabilizing the enzyme in the dimeric conformation.
Figure 1. Effects of CTZ on PFK activity in the absence and presence of aldolase. PFK activity was assessed as described under
‘‘Materials and Methods’’ in the absence and in the presence of 1 lg/mL aldolase. Panel A: Dose-response curve of CTZ effects on
PFK activity in the absence (control, filled circles) and in the presence of 1 lg/mL aldolase (empty circles). Solid line shown for
the absence of aldolase was obtained fitting Eq. (2) to the experimental data. Solid line for the presence of aldolase is the linear
regression of the plotted data. Panel B: Relative remaining PFK activity in the presence of 50 lM CTZ assessed with the concen-
trations of PFK indicated on the abscissa. The experiments were performed in the absence (control, filled circles) and in the pres-
ence of 1 lg/mL aldolase (empty circles). All plotted data are mean 6 standard errors of, at least, four independent experiments (n
437ASSOCIATION OF PFK AND ALDOLASE
However, these PFK dimers bound to aldolase maintain a cata-
lytic activity similar to that observed for the tetramers (10).
This fact could explain the lack of PFK inhibition by CTZ in
the presence of aldolase, as PFK dimers formed due to the
effects of CTZ would bind to aldolase and thus maintain their
catalytic activity. To corroborate this hypothesis, we evaluated
the effects of CTZ on PFK activity at different concentrations
of the enzyme.
It has been reported that the equilibrium between PFK
dimers and tetramers is directly affected by the concentration of
the enzyme; the more concentrated the enzyme is, the more sta-
ble tetramers are (6, 11). In fact, when increasing the concentra-
tion of PFK in the reaction medium, a progressive decrease of
the inhibition promoted by CTZ is observed. For example, in
the presence of 1.5 lg/mL PFK, no significant effect of CTZ
was observed (Fig. 1B, filled circles). Moreover, aldolase was
able to abrogate the effects of CTZ on PFK at all PFK concen-
trations used (Fig. 1B, empty circles). Furthermore, we deter-
mined that 1 mol of aldolase monomer per mol of PFK mono-
mer is required for aldolase to protect PFK from the inhibitory
effects of CTZ. This can be observed in Fig. 2, which shows
that a stoichiometry of 2 PFK monomers per aldolase monomer
does not alter the inhibition of PFK by CTZ. However, when
aldolase is present at a concentration proportionally equal to or
in excess of that of PFK, full protection from CTZ is observed
(Fig. 2). These data support the hypothesis that aldolase binds
to the PFK dimers formed due to the effects of CTZ, turning
these dimers into an active dimeric conformation and thus pre-
venting the inhibitory effects of the drug.
Structural Evidences for the Counteraction of
CTZ-Induced PFK Inhibition by Aldolase
Evidences for the interaction between PFK and aldolase
were assessed through two distinct techniques: intrinsic fluores-
cence emission spectroscopy and light scattering. We have effi-
ciently applied these methods to evaluate the transition between
PFK dimers and tetramers (4–6, 8, 11, 18, 22, 23, 29, 35, 36).
Intrinsic fluorescence measurements show a slight red-shift of
PFK intrinsic fluorescence emission spectrum in the presence of
aldolase (Fig. 3A, cf. the black line for PFK with the green line
for PFK in the presence of aldolase). This effect becomes clear
calculating the center of mass of these spectra. The center of
mass of the intrinsic fluorescence emission spectra of PFK alone
is 338.2 6 0.4 nm (mean 6 standard error of four independent
experiments; n 5 4). In the presence of aldolase, the calculated
center of mass of PFK intrinsic fluorescence emission spectra
shifts to 341.6 6 0.5 nm (P \ 0.05 comparing to control in the
absence of aldolase; Student’s t-test; n 5 4). This later value is
not different from the center of mass of PFK intrinsic fluores-
cence spectra in the presence of CTZ or aldolase and CTZ
(341.3 6 0.4 nm and 341.4 6 0.4 nm, respectively). This red-
shift observed is characteristic of the dissociation of the tet-
ramers of the kinase into dimers, as well documented in previous
publications (4–6, 8, 11, 18, 22, 23, 29, 35, 36). It cannot be due
to the simple interference of aldolase intrinsic fluorescence emis-
sion since the signal for this emission is negligible when com-
pared to PFK signal (Fig. 3A, cf. the red line for aldolase with
the black line for PFK). Moreover, the presence of 50 lM CTZ,
which has been demonstrated to dissociate PFK tetramers into
dimers (23, 29), induced a similar effect on PFK intrinsic fluo-
rescence emission spectrum (Fig. 3A, yellow line). This pattern
is not modified in the simultaneous presence of aldolase and
CTZ (Fig. 3A, blue line). These results suggest that both aldol-
ase and CTZ stabilize the dimeric conformation of PFK.
To corroborate this hypothesis, we evaluated the ability of
the proteins to scatter light, which is proportional to the dimen-
sion of the protein particles in solution. As expected, PFK scat-
ters more light than aldolase (Fig. 3B, cf. the black line, for
PFK, with the red line, for aldolase), which is compatible with
the higher molecular weight of the former comparing to the
later ( ~ 340 kDa for PFK tetramers vs. ? 180 kDa for aldolase
tetramers). CTZ strongly promotes the attenuation of the light
scattered by PFK (Fig. 3B, yellow line), which is indicative of
the dissociation of the enzyme. On the other hand, CTZ almost
did not affected light scattering by aldolase (Fig. 3B, magenta).
The simultaneous presence of PFK and aldolase in the medium
promotes a pattern of light scattering (Fig. 3B, green line) that
is intermediate between those observed with PFK alone and in
the presence of CTZ. This is indicative that the protein particles
under this conditions present an intermediate size between the
Figure 2. Reversal of CTZ-induced inhibition of PFK by aldol-
ase. PFK activity was assessed as described under ‘‘Materials
and Methods’’ in the presence of 1 lg/mL PFK and increasing
concentrations of aldolase. The molar ratio was calculated con-
sidering PFK monomer presenting 85 kDa and aldolase mono-
mer 45 kDa. Experiments were performed in the absence and in
the presence of 50 lM CTZ as indicated. Bars are mean 6
standard errors of, at least, four independent experiments (n 5
4). *P \ 0.05 comparing with control experiments in the ab-
sence of CTZ (Student’s t-test).
438MARCONDES ET AL.
tetrameric and dimeric PFK conformations. Moreover, the si-
multaneous presence of PFK and aldolase produces protein par-
ticles that scatter more light than aldolase alone and less than
PFK alone. Together with the results described above, this
result suggests that PFK and aldolase might associate into heter-
otetramers formed by a PFK and an aldolase dimer. The ability
of PFK and aldolase to generate such an heterotetramer have
been demonstrated before, using other techniques (10). This het-
erotetramer, presenting ?260 kDa, would be compatible with
the light scattering pattern observed in the simultaneous pres-
ence of PFK and aldolase (Fig. 3B, green line). This heterote-
tramer does not dissociate in the presence of CTZ, since the
drug did not affect the light scattering pattern of the associated
proteins (Fig. 3B, blue line). These results suggest that CTZ is
not able to inhibit PFK in the presence of aldolase because it is
unable to dissociate the active heterotetrameric enzyme complex
formed by the two enzymes.
CTZ Affects the Kinetic Parameters of PFK, but not in
the Presence of Aldolase
The kinetic parameters for PFK activation by its substrates
were evaluated in the presence of CTZ and aldolase. As previ-
ously reported (29), CTZ alters the effects of the substrates on
enzyme kinetics (Figs. 4A and 4C), decreasing the maximal ve-
locity and the affinity of PFK for F6P and ATP at the catalytic
site, while increasing the affinity of the enzyme for ATP at its
inhibitory site (Table 1). In the presence of aldolase, the affinity
of PFK for its substrate at the catalytic site is also lower when
compared to the control in the absence of aldolase (Table 1).
This occurs in spite of the fact that the maximal velocity of
PFK and the affinity of ATP inhibitory site are not altered by
aldolase (Table 1). On the other hand, in the presence of aldol-
ase, CTZ presented no effect on PFK activation by its substrates
(Figs. 4B and 4D; Table 1). The diminished affinity for ATP
and F6P at PFK catalytic site is characteristic of the dimeric
conformation of the enzyme (1) and is observed in the presence
of both, CTZ or aldolase.
Allosteric Modulators of PFK Interfere with the
Counteraction of PFK Inhibition by Aldolase
We evaluated the effects of CTZ on PFK activity in the pres-
ence of other modulators of the enzyme’s quaternary structure,
in both the absence and the presence of aldolase. For this, we
used ADP and fructose-2,6-bisphosphate (F2,6BP), two stabil-
izers of the tetrameric structure of the enzyme, and citrate and
lactate, two stabilizers of the enzyme dimers (4–6, 8). In the ab-
sence of aldolase (Fig. 5A), both stabilizers of PFK tetramers
attenuated the inhibitory effects of CTZ. ADP was much more
efficient than F2,6BP in both activating PFK and protecting the
enzyme against the inhibitory effects of 50 lM CTZ; ADP pro-
motes a fourfold stimulation of the enzyme activity and abro-
gates the effects of CTZ. F2,6BP increased the enzyme activity
by 30% and attenuated the enzyme inhibition to 25%, versus
50% for the control (Fig. 5A). The stimulatory effects of F2,6BP
are more pronounced when the enzyme is inhibited by ATP (5,
6), which is not the case since the ATP concentration in this
experiment is not inhibitory. Therefore, since these experiments
were performed in the presence of 1 mM ATP, which is not in-
hibitory for the enzyme (5, 6), it is expected that ADP would be
more effective than F2,6BP in activating the enzyme and, thus,
in counteracting the inhibitory effects of CTZ. When these
experiments were performed in the presence of citrate, which
Figure 3. Structural evaluation of PFK and aldolase in the ab-
sence and the presence of CTZ. Panel A: intrinsic fluorescence
emission of the enzymes was evaluated as described under
‘‘Materials and Methods’’ exciting the samples at 280 nm and the
fluorescence emission was scanned from 300 to 400 nm and rep-
resented as arbitrary units (A.U.). The curves are representative
spectra of four independent experiments. Panel B: light scattering
experiments were performed as described under Materials and
Methods, exciting the samples at 510 nm and recording the light
scattered from 500 to 510 nm, which is represented as arbitrary
units (A.U.). The curves are representative light results of four in-
dependent experiments. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
439ASSOCIATION OF PFK AND ALDOLASE
Figure 4. Effects of aldolase and CTZ on the modulation of PFK by its substrates. PFK activity was assessed as described under
Materials and Methods in the absence and in the presence of 50 lM CTZ, as indicated. Panels A and D are, respectively, the ATP
and F6P curves assayed in the absence of aldolase. Panels B and D are, respectively, the ATP and F6P curves assayed in the pres-
ence of 1 lg/mL aldolase. Plotted values as mean 6 standard error of four independent experiments. Solid lines are the results of
the adjust of Eq. (5) (panels A and C) and Eq. (6) (panels B and D) to the experimental plotted data.
Kinetic parameters for PFK modulation by its substrates
Control50 lM CTZ1 lg/mL aldolase1 lg/mL aldolase 1 50 lM CTZ
0.31 6 0.03
1.1 6 0.2
15.2 6 1.7
3.2 6 0.3
1.7 6 0.2
16.6 6 1.4
0.30 6 0.03
1.1 6 0.1
0.79 6 0.06a
2.0 6 0.3a
11.6 6 1.3a
1.5 6 0.2a
1.2 6 0.2
12.2 6 1.3a
0.49 6 0.03a
2.2 6 0.2a
0.75 6 0.05a
1.8 6 0.2a
15.8 6 1.6
2.9 6 0.3
1.5 6 0.2
16.5 6 1.6
0.50 6 0.04a
1.7 6 0.3a
0.75 6 0.07
1.9 6 0.3
15.8 6 1.5
3.0 6 0.3
1.6 6 0.2
16.4 6 1.6
0.51 6 0.05
1.7 6 0.2
The parameters were calculated as described under ‘‘Materials and Methods’’ section, using Eq. (5) to calculate the parameter for ATP curve and equation
(6) for F6P curve.
aP \ 0.05 compared to control (Student’s t-test).
440 MARCONDES ET AL.
inhibits the enzyme, favoring the formation of dimers (4), the
effects of CTZ were enhanced, suggesting an additive effect
(Fig. 5A). On the other hand, lactate was not able to reinforce
the inhibitory effects of CTZ, despite the fact that both CTZ and
lactate inhibit the enzyme by stabilizing the dimeric conforma-
tion (4, 8, 23, 29). Curiously, 10 mM lactate alone resulted in
52% 6 3% inhibition of PFK activity, which was equivalent to
the effects of 50 lM CTZ (48% 6 2%) or lactate and CTZ to-
gether (51% 6 5%). Two hypotheses may explain this phenom-
enon: a) lactate and CTZ bind to the same site at the enzyme; or
b) the binding of one blocks the effect of the other.
In the presence of 1 lg/mL aldolase (a molar ratio of 1:2
PFK:aldolase), the above pattern changes. ADP and F2,6BP did
not activate PFK (Fig. 5B), and CTZ did not cause the typical
inhibitory effects in the presence of the two PFK activators
(Fig. 5B). These results can be explained by the formation of
active PFK dimers in the presence of aldolase. Since ADP and
F2,6BP activate PFK, favoring the formation of tetramers, their
effects were not seen when active PFK dimers were stabilized
by aldolase. In a reciprocal way, since these dimers were active,
CTZ was not able to inhibit the enzyme because the drug pro-
motes the dissociation of the active tetramers into inactive
dimers. In conclusion, ADP, F2,6BP and CTZ did not interfere
with the formation of active PFK dimers induced by aldolase.
On the other hand, the presence of aldolase did not prevent
PFK inhibition by citrate or lactate (Fig. 5B). The inhibitory
effects of citrate occur to the same extent in the absence or in
the presence of aldolase. However, lactate is a more effective
inhibitor of PFK in the presence of aldolase than in its absence
(Fig. 5). Moreover, in the presence of aldolase, citrate enhanced
the inhibitory effects of CTZ on PFK, while in the presence of
lactate, there was no additional effect of CTZ on PFK inhibition
(Fig. 5B), mirroring the results observed in the absence of al-
dolase (Fig. 5A). In the presence of aldolase, the inhibitory
effects of 10 mM lactate were more pronounced than in the ab-
sence of the enzyme (50% vs. 75% inhibition in the absence vs.
presence of aldolase, respectively). The fact that the presence of
aldolase did not interfere with the inhibition promoted by citrate
but accentuated the inhibitory action of lactate suggests that
demonstrated that both lactate and citrate stabilize the dimeric
conformation of PFK (4, 8), as does aldolase (10). Therefore, a
possible explanation for the fact that aldolase potentiates the in-
hibitory effects of lactate but not of citrate is that lactate binds
preferentially to PFK dimers. Because PFK dimers are already
formed in the presence of aldolase, lactate would bind more
easily and thus be more effective. Moreover, these results an-
swer the question raised above concerning the lack of additive
effects for lactate and CTZ; we can infer that the binding of
lactate to PFK blocks the effects of CTZ.
PFK Stimulates Aldolase Activity
CTZ has also been shown to affect the activities of other
glycolytic enzymes, such as hexokinase (26, 35) and aldolase
(20). Therefore, we decided to evaluate the effects of CTZ on
aldolase activity in the absence and the presence of PFK. CTZ
results in the dose-dependent inhibition of aldolase in the ab-
sence and in the presence of PFK (Fig. 6A). Surprisingly, the
presence of 1 lg/mL PFK in the reaction medium resulted in a
twofold stimulation of aldolase activity, which persisted in the
presence of all concentrations of CTZ tested (Fig. 6A). To our
knowledge, this is the first time that stimulation of aldolase ac-
tivity by PFK has been reported. Despite stimulating aldolase
activity, PFK is not able to prevent CTZ-induced inhibition of
aldolase. The effects of CTZ on aldolase reached a maximal in-
hibition of 98% 6 2% and followed a parallel pattern under
Figure 5. Influence of allosteric modulators of PFK on CTZ-
induced inhibition of PFK in the absence and presence of aldol-
ase. PFK activity was assessed as described under ‘‘Materials
and Methods’’ in the absence and in the presence of 50 lM
CTZ, as indicated. The allosteric modulators of PFK were
added at the concentrations indicated on the abscissa, in the ab-
sence (Panel A) and in the presence of 1 lg/mL aldolase (Panel
B). Bars are mean 6 standard errors of, at least, four independ-
ent experiments (n 5 4). *P \ 0.05 comparing with control
experiments in the absence of CTZ (Student’s t-test). #P \ 0.05
comparing with the respective bar where no allosteric modulator
was added (‘‘no addition’’; Student’s t-test).
441 ASSOCIATION OF PFK AND ALDOLASE
both conditions, presenting I0.5values of 11 6 1 lM and 10 6
1 lM in the absence and presence of PFK, respectively. These
values are not statistically different, suggesting that the presence
of PFK does not affect the inhibition of aldolase by CTZ. The
inhibitory effects of CTZ on aldolase activity were not influ-
enced by the concentration of the enzyme in the reaction me-
dium (Fig. 6B, cf. filled circles with filled triangles for control
and for the presence of 50 lM CTZ, respectively). The presence
of the drug resulted in ?95% inhibition of aldolase activity in
the absence and in the presence of PFK for all concentrations
of aldolase tested (Fig. 6B). However, comparing the effects of
CTZ on aldolase activity in the absence and the presence of
PFK, it is clear that in the latter condition aldolase activity was
doubled when compared to the former (Fig. 6B, cf. filled trian-
gles and empty triangles for the absence and the presence of
To evaluate the activator property of PFK on aldolase, we
assessed the aldolase activity in the presence of increasing con-
centrations of PFK. Aldolase activity increased with increasing
concentrations of PFK in a dose-dependent manner (Fig. 7),
reaching a maximal activation of 95% 6 8% and an activation
constant (Ka) of 0.45 6 0.03 lg/mL (?5.3 nM for PFK mono-
mers). These parameters, obtained in the presence of 50 lM
CTZ, are not statistically different from the control (93% 6 9%
activation and Ka 5 0.48 6 0.05 lg/mL; P [ 0.05, Student’s t-
test). The maximal activation of aldolase by PFK was reached in
the presence of 1 lg/mL PFK (Fig. 7), representing a molar ratio
of 2:1 (aldolase:PFK, based on the monomers of both enzymes).
The kinetic parameters for the modulation of aldolase by its
substrate F1,6BP were evaluated in the absence and in the pres-
ence of CTZ and PFK. CTZ inhibited aldolase in the presence
Figure 6. Effects of CTZ on aldolase activity in the absence and presence of PFK. Aldolase activity was assessed in the absence
and in the presence of 1 lg/mL PFK, as described in the ‘‘Materials and Methods’’ section. Panel A: Dose-response curve of CTZ
effects on aldolase activity in the absence (control, filled triangles) and in the presence of 1 lg/mL PFK (empty triangles). Solid
lines were obtained by fitting Eq. (2) to the experimental data. Panel B: aldolase activity assessed using the concentrations of aldol-
ase indicated on the abscissa. The experiments were performed with no further additions (control, filled circles), in the presence of
1 lg/mL PFK (empty circles), in the presence of 50 lM CTZ (filled triangles) and in the presence of 1 lg/mL PFK and 50 lM
CTZ (empty triangles). Solid lines are the result of the linear regression of the plotted data, and dashed lines are the extrapolation
of the linear regression results. All plotted data are mean 6 standard errors of at least four independent experiments (n 5 4).
Figure 7. Effects of PFK on aldolase activity in the absence
and presence of 50 lM CTZ. Aldolase activity in the presence
of 1 lg/mL aldolase and the concentrations of PFK indicated
on the abscissa was assessed as described in the ‘‘Materials and
Methods’’ section. Experiments were performed in the absence
(filled circles) and in the presence of 50 lM CTZ (empty
circles). Plotted values are mean 6 standard errors of at least
four independent experiments (n 5 4). Solid lines were
obtained fitting Eq. (7) to the plotted data.
442MARCONDES ET AL.
of all concentrations of F1,6BP tested, in the absence and in the
presence of PFK (Fig. 8). This effect resulted in a decrease of
the enzyme maximal velocity and affinity for its substrate (Ta-
ble 2). On the other hand, PFK promoted an increase in both
the aldolase maximal velocity and the affinity of F1,6BP. Tak-
ing another point of view, the effects of CTZ on aldolase in the
presence of PFK are attenuated, when compared to the effects
of the drug in the absence of the kinase (Table 2). These results
do not suggest that PFK protects aldolase against CTZ inhibi-
tion, but clearly demonstrate that the interaction between the
two enzymes upregulates aldolase increasing both, its maximal
velocity and its affinity for the substrate.
CTZ has been described as a potential antineoplastic drug
(20, 23, 27, 28, 37, 38). Its antineoplastic properties are associ-
ated with its ability to decrease glucose consumption and
energy metabolism in tumor cells (27, 28). There are several
proposed targets for the action of CTZ on cell metabolism, and
many of these proposed targets are involved in the glycolytic
pathway (38, 39). We have previously demonstrated that this
drug decreases the viability of breast cancer cells by inhibiting
the glycolytic pathway (20) and that this inhibition is probably
due to a direct effect of CTZ on PFK (23, 29). The data pre-
sented here add novel information concerning the ability of
CTZ to affect glycolytic enzymes and, therefore, cancer cell
metabolism. It appears that aldolase is able to prevent the inhib-
itory effect of CTZ on the major glycolytic regulatory enzyme,
PFK. However, CTZ inhibits aldolase to a greater degree (98%
inhibition for aldolase vs. 70% for PFK) and more efficiently
(I0.5 5 11 6 1 lM and 28 6 2 lM for aldolase and PFK,
respectively). This means that, despite the protection against
CTZ-induced PFK inhibition promoted by aldolase, CTZ still
decreases the glycolytic flux through inhibition of the aldolase
itself, the next enzyme in the pathway following PFK.
The protection against the inhibitory effects of CTZ on PFK
promoted by aldolase would be expected. Because CTZ induces
the dissociation of active PFK tetramers into inactive dimers
(23, 29), aldolase, which binds to PFK dimers and renders them
active (10), should counteract the effects of CTZ on PFK. How-
ever, another inhibitory modulator of PFK, citrate, disrupts the
Figure 8. Effects of PFK and CTZ on the modulation of aldolase by its substrate. Aldolase activity was assessed as described
under Materials and Methods in the absence and in the presence of 50 lM CTZ, as indicated. Panels A: control experiments in the
absence of PFK. Panels B: aldolase activity in the presence of 1 lg/mL PFK. Plotted values as mean 6 standard error of four inde-
pendent experiments. Solid lines are the results of the adjust of Eq. (6) to the experimental plotted data.
Kinetic parameters for aldolase modulation by F1,6BP
Control 50 lM CTZ1 lg/mL PFK1 lg/mL PFK 1 50 lM CTZ
8.7 6 0.9
68.1 6 4.2
1.4 6 0.2
1.6 6 0.2a
103.4 6 9.7a
2.0 6 0.2a
17.9 6 1.8a
34.1 6 3.8a
0.9 6 0.3a
4.1 6 0.5b,c
89.1 6 3.9b,c
2.1 6 0.2b,c
The parameters were calculated as described under ‘‘Materials and Methods’’ section.
aP \ 0.05 compared to control (Student’s t-test).
bP \ 0.05 compared to 50 lM CTZ.
cP \ 0.05 compared to 1 lg/mL aldolase.
443ASSOCIATION OF PFK AND ALDOLASE
protection conferred by aldolase against CTZ-induced inhibition
of PFK. In the presence of citrate, CTZ is a more effective in-
hibitor of PFK than in the absence of the metabolite. In tumor
cells, cytosolic citrate levels are elevated because of the need
for fatty acid and cholesterol biosynthesis (40). Thus, CTZ
might inhibit PFK in these cells due to the high levels of ci-
An intriguing finding in the present work is the stimulatory
property of PFK over aldolase. The ability of these enzymes to
associate generating diverse heterooligomeric structures has
been reported (10). However, the ability of PFK to increase al-
dolase activity has not been hitherto published. The fact that
PFK augments the affinity of aldolase for its substrate strongly
supports the hypothesis that the kinase, once associated to aldol-
ase, stimulates its activity. This is an evidence for the channel-
ing of the glycolytic metabolism, as have been previously pro-
posed (13–15). Actually, this is not a classical channeling since
PFK-bound aldolase is able to bind F1,6BP directly from the
medium and not necessarily those formed by PFK catalysis.
Nonetheless, the association of PFK and aldolase confers a cata-
lytical advantage for both enzymes, stabilizing the active di-
meric conformation of the former and stimulating the later.
This work was supported by grants from the Fundac ¸a ˜o Carlos
Chagas Filho de Amparo a Pesquisa do Estado do Rio de
Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento
Cientı ´fico e Tecnolo ´gico (CNPq), Programa de Nu ´cleos de
Excele ˆncia (PRONEX), and Conselho de Aperfeic ¸oamento de
Pessoal de Nı ´vel Superior (CAPES).
1. Sola-Penna, M., Da Silva, D., Coelho, W. S., Marinho-Carvalho, M. M.,
and Zancan, P. (2010) Regulation of mammalian muscle type 6-phos-
phofructo-1-kinase and its implication for the control of the metabolism.
IUBMB Life 62, 791–796.
2. Hesterberg, L. K. and Lee, J. C. (1980) Sedimentation study of a cata-
lytically active form of rabbit muscle phosphofructokinase at pH 8.55.
Biochemistry 19, 2030–2039.
3. Hesterberg, L. K., Lee, J. C., and Erickson, H. P. (1981) Structural
properties of an active form of rabbit muscle phosphofructokinase. J.
Biol. Chem. 256, 9724–9730.
4. Marinho-Carvalho, M. M., Costa-Mattos, P. V., Spitz, G. A., Zancan,
P., and Sola-Penna, M. (2009) Calmodulin upregulates skeletal muscle
6-phosphofructo-1-kinase reversing the inhibitory effects of allosteric
modulators. Biochim. Biophys. Acta. 1794, 1175–1180.
5. Zancan, P., Almeida, F. V., Faber-Barata, J., Dellias, J. M., and Sola-
Penna, M. (2007) Fructose-2,6-bisphosphate counteracts guanidinium
chloride-, thermal-, and ATP-induced dissociation of skeletal muscle
key glycolytic enzyme 6-phosphofructo-1-kinase: a structural mecha-
nism for PFK allosteric regulation. Arch. Biochem. Biophys. 467, 275–
6. Zancan, P., Marinho-Carvalho, M. M., Faber-Barata, J., Dellias, J. M.,
and Sola-Penna, M. (2008) ATP and fructose-2,6-bisphosphate regulate
skeletal muscle 6-phosphofructo-1-kinase by altering its quaternary
structure. IUBMB Life 60, 526–533.
7. Hesterberg, L. K. and Lee, J. C. (1982) Self-association of rabbit mus-
cle phosphofructokinase: effects of ligands. Biochemistry 21, 216–222.
8. Leite, T. C., Da Silva, D., Coelho, R. G., Zancan, P., and Sola-Penna,
M. (2007) Lactate favours the dissociation of skeletal muscle 6-phos-
phofructo-1-kinase tetramers down-regulating the enzyme and muscle
glycolysis. Biochem. J. 408, 123–130.
9. Vertessy, B. G., Orosz, F., Kovacs, J., and Ovadi, J. (1997) Alternative
binding of two sequential glycolytic enzymes to microtubules. Molecu-
lar studies in the phosphofructokinase/aldolase/microtubule system. J.
Biol. Chem. 272, 25542–25546.
10. Rais, B., Ortega, F., Puigjaner, J., Comin, B., Orosz, F., Ovadi, J., and
Cascante, M. (2000) Quantitative characterization of homo- and hetero-
associations of muscle phosphofructokinase with aldolase. Biochim. Bio-
phys. Acta. 1479, 303–314.
11. Marinho-Carvalho, M. M., Zancan, P., and Sola-Penna, M. (2006) Mod-
ulation of 6-phosphofructo-1-kinase oligomeric equilibrium by calmodu-
lin: formation of active dimers. Mol. Genet. Metab. 87, 253–261.
12. Masters, C. (1984) Interactions between glycolytic enzymes and compo-
nents of the cytomatrix. J. Cell Biol. 99, 222s–225s.
13. Srere, P. A. (1987) Complexes of sequential metabolic enzymes. Annu.
Rev. Biochem. 56, 89–124.
14. Srere, P. A.. and Ovadi, J. (1990) Enzyme-enzyme interactions and their
metabolic role. FEBS Lett. 268, 360–364.
15. Srere, P. (1994) Complexities of metabolic regulation. Trends Biochem.
Sci. 19, 519–520.
16. Alves, G. G. and Sola-Penna, M. (2003) Epinephrine modulates cellular
distribution of muscle phosphofructokinase. Mol. Genet. Metab. 78,
17. Coelho, W. S., Costa, K. C., and Sola-Penna, M. (2007) Serotonin stim-
ulates mouse skeletal muscle 6-phosphofructo-1-kinase through tyro-
sine-phosphorylation of the enzyme altering its intracellular localization.
Mol. Genet. Metab. 92, 364–370.
18. Da Silva, D., Zancan, P., Coelho, W. S., Gomez, L. S., and Sola-Penna,
M. (2010) Metformin reverses hexokinase and 6-phosphofructo-1-kinase
inhibition in skeletal muscle, liver and adipose tissues from streptozoto-
cin-induced diabetic mouse. Arch. Biochem. Biophys. 496, 53–60.
19. El-Bacha, T., de Freitas, M. S., and Sola-Penna, M. (2003) Cellular
distribution of phosphofructokinase activity and implications to meta-
bolic regulation in human breast cancer. Mol. Genet. Metab. 79, 294–
20. Meira, D. D., Marinho-Carvalho, M. M., Teixeira, C. A., Veiga, V. F.,
Da Poian, A. T., Holandino, C., de Freitas, M. S., and Sola-Penna, M.
(2005) Clotrimazole decreases human breast cancer cells viability
through alterations in cytoskeleton-associated glycolytic enzymes. Mol.
Genet. Metab. 84, 354–362.
21. Real-Hohn, A., Zancan, P., Da Silva, D., Martins, E. R., Salgado, L. T.,
Mermelstein, C. S., Gomes, A. M., and Sola-Penna, M. (2010) Filamen-
tous actin and its associated binding proteins are the stimulatory site for
6-phosphofructo-1-kinase association within the membrane of human
erythrocytes. Biochimie 92, 538–544.
22. Spitz, G.A., Furtado, C. M., Sola-Penna, M., and Zancan, P. (2009) Ac-
etylsalicylic acid and salicylic acid decrease tumor cell viability and
glucose metabolism modulating 6-phosphofructo-1-kinase structure and
activity. Biochem. Pharmacol. 77, 46–53.
23. Zancan, P., Rosas, A. O., Marcondes, M. C., Marinho-Carvalho, M. M.,
and Sola-Penna, M. (2007) Clotrimazole inhibits and modulates heterol-
ogous association of the key glycolytic enzyme 6-phosphofructo-1-ki-
nase. Biochem. Pharmacol. 73, 1520–1527.
24. Zancan, P., Sola-Penna, M., Furtado, C. M., and Da Silva, D. (2010)
Differential expression of phosphofructokinase-1 isoforms correlates
with the glycolytic efficiency of breast cancer cells. Mol. Genet. Metab.
25. Gatenby, R. A. and Gillies, R. J. (2004) Why do cancers have high aer-
obic glycolysis? Nat. Rev. Cancer 4, 891–899.
444MARCONDES ET AL.
26. Penso, J. and Beitner, R. (1998) Clotrimazole and bifonazole detach
hexokinase from mitochondria of melanoma cells. Eur. J. Pharmacol.
27. Penso, J. and Beitner, R. (2002) Detachment of glycolytic enzymes
from cytoskeleton of Lewis lung carcinoma and colon adenocarcinoma
cells induced by clotrimazole and its correlation to cell viability and
morphology. Mol. Genet. Metab. 76, 181–188.
28. Penso, J. and Beitner, R. (2002) Clotrimazole decreases glycolysis and
the viability of lung carcinoma and colon adenocarcinoma cells. Eur. J.
Pharmacol. 451, 227–235.
29. Marcondes, M. C., Sola-Penna, M., and Zancan, P. (2010) Clotrimazole
potentiates the inhibitory effects of ATP on the key glycolytic enzyme
6-phosphofructo-1-kinase. Arch. Biochem. Biophys. 497, 62–67.
30. Maia, J. C. C., Gomes, S. L., Juliani, M. H., and Morel, C. M. (1983)
Preparation of [y- 32 P] and [a- 32 P]-nucleoside triphosphate, with
high specific activity. In: Genes and Antigenes of Parasites: a Labora-
tory Manual (Morel, C. M., ed.), Vol. 1. pp. 146–157, Fiocruz, Brazil.
31. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)
Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,
32. Sola-Penna, M., dos Santos, A. C., Alves, G. G., El-Bacha, T., Faber-
Barata, J., Pereira, M. F., Serejo, F. C., Da Poian, A. T., and Sorenson,
M. (2002) A radioassay for phosphofructokinase-1 activity in cell
extracts and purified enzyme. J. Biochem. Biophys. Methods 50, 129–
33. Zancan, P. and Sola-Penna, M. (2005) Calcium influx: a possible role
for insulin modulation of intracellular distribution and activity of 6-
phosphofructo-1-kinase in human erythrocytes. Mol. Genet. Metab. 86,
34. Zancan, P. and Sola-Penna, M. (2005) Regulation of human erythrocyte
metabolism by insulin: cellular distribution of 6-phosphofructo-1-kinase
and its implication for red blood cell function. Mol. Genet. Metab. 86,
35. Leite, T. C., Coelho, R. G., Silva, D. D., Coelho, W. S., Marinho-Car-
valho, M. M., and Sola-Penna, M. (2011) Lactate downregulates the
glycolytic enzymes hexokinase and phosphofructokinase in diverse tis-
sues from mice. FEBS Lett. 585, 92–98.
36. Faber-Barata, J. and Sola-Penna, M. (2005) Opposing effects of two
osmolytes—trehalose and glycerol—on thermal inactivation of rabbit
muscle 6-phosphofructo-1-kinase. Mol. Cell Biochem. 269, 203–207.
37. Khalid, M. H., Tokunaga, Y., Caputy, A. J., and Walters, E. (2005) In-
hibition of tumor growth and prolonged survival of rats with intracranial
gliomas following administration of clotrimazole. J. Neurosurg. 103,
38. RodrI`guez-EnrI`quez, S., MarI`n-Hern?ndez, A., Gallardo-PE`rez, J. C.,
CarreO´o-Fuentes, L., and Moreno-Sa ´nchez, R. (2009) Targeting of can-
cer energy metabolism. Mol. Nutr. Food Res. 53, 29–48.
39. Moreno-Sanchez, R., Rodriguez-Enriquez, S., Marin-Hernandez, A., and
Saavedra, E. (2007) Energy metabolism in tumor cells. FEBS J. 274,
40. Costello, L. C. and Franklin, R. B. (2005) ‘Why do tumour cells glyco-
lyse?’: from glycolysis through citrate to lipogenesis. Mol. Cell. Bio-
chem. 280, 1–8.
445ASSOCIATION OF PFK AND ALDOLASE