Systems Biology Approaches to Cancer Energy Metabolism - Systems Biology of Metabolic and Signaling Networks - page 221

Table 9.3 Ratios of GLUT, HK, and PFK isoforms in HeLa cells exposed to normoxia or hypoxia

Normoxia Hypoxia

Isoform % of band intensity Fraction % of band intensity Fraction

GLUT1 41 0.97 108 0.99

GLUT3 1.3 0.03 1 0.01

GLUT total

42.3 109

HKI 10.3 0.36 10.8 0.1

HKII 18.3 0.64 98 0.9

HK total 28.6 108.8

PFK-C 63 0.95 88 0.46

PFK-L 3 0.05 104 0.54

PFK total 66 192

in which Glu out and Glu in are the extra- and intracellular glucose concentrations,
K Gluout1 and K Gluout2 are the affinities for extracellular glucose of each GLUT
isoform, K eq is the equilibrium constant of the reaction, V mf is the maximal velocity
in the forward reaction determined in the cells, and f 1 and f 2 are the proportion of
each isoform determined by western blot analysis (Fig. 9.2 , Table 9.3 ).
The HK rate was formulated to represent a double random-bisubstrate
Michaelis-Menten equation ( 9.2 ), in which A and B are Glu in and ATP, and P
and Q are G6P and ADP, respectively; Ka 1 and Ka 2 represent the affinity constants
for Glu in of each isoform whereas Kb is the affinity for ATP which did not change in
both isoforms; Kp and Kq are the affinity constants for the corresponding products.
The rate equation for PFK-1 ( 9.3 ) obeys a double concerted transition model of
Monod, Wyman, and Changeux for exclusive ligand binding (F6P, activators,
inhibitors) together with mixed-type activation (F2,6BP or AMP or Pi) (Moreno-
S´nchez et al. 2012 ) and simple Michaelis-Menten terms for ATP and the reverse
reaction. ATP (at high concentrations) and citrate are the allosteric inhibitors. L 1
and L 2 are the allosteric transition constants; Ka F2,6BP1 and Ka F2,6BP2 are the
activation constants for F2,6BP; Ki CIT1 , Ki CIT2 Ki ATP1 , and Ki ATP2 are the inhibition
constants for citrate and ATP; 2 are the factors by which K F6P

( K F6P1 and K F6P2 ) and V max change, respectively, when an activator is bound to the
active enzyme form (R conformation in the Monod model).
Each isoform ratio was determined (Fig. 9.2 ) considering that the antibodies
have high specificity for their respective isoforms as previously determined
(Rodr´guez-Enr´quez et al. 2009 ; Moreno-S´nchez et al. 2012 ) but assuming that
they have similar specificity and detection sensitivity. The sum of the band
intensities for both isoforms was considered the total protein content from which
the corresponding fraction of each isoform was calculated (Table 9.3 ). These values
are denoted by the coefficient f in the corresponding rate equations.

α 1, α 2 and β 1, β