SUPPORTING INFORMATION
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ATP Production from PCr Breakdown
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The rate of ATP production from the breakdown of PCr through the CK reaction (ATP CK,
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mM.min-1) was calculated from the change in PCr for each time point of the exercise period
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(Kemp and Radda, 1994):
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ATPCK = dPCr/dt
[A1]
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ATP Production from Oxidative Phosphorylation
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Based on the hyperbolic relationship between the oxidative ATP production rate (Q, mM.min -1)
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and free cytosolic ADP concentration ([ADP]), the rate of mitochondrial ATP production
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(ATPOX) was calculated as follows:
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ATPOX = Qmax/(1+Km/[ADP])
[A2]
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in which Km (the [ADP] at half-maximal oxidation rate) is ~30 μM in skeletal muscle (Kemp
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and Radda, 1994) and Qmax is the maximal rate of oxidative ATP production. Qmax (in mM.min-
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1
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and [ADP] was measured at the end of exercise:
) was calculated using the initial rate of PCr synthesis (V recov PCr) during the recovery period
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Qmax = Vrecov PCr (1+Km/[ADP]end)
[A3]
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ATP Production from Anaerobic Glycolysis
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Throughout the exercise period, glycogen breakdown to pyruvate and lactate, proton efflux,
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buffering capacity, protons produced by oxidative phosphorylation and the consumption of
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protons by the CK reaction lead to changes in intramuscular pH (Kemp and Radda, 1994).
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Assuming that the glycogenolytic production of 1 mole of H+, when coupled to ATP hydrolysis,
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yields 1.5 moles of ATP, the rate of ATP production from anaerobic glycolysis (ATPGLY) can be
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deduced from the total number of protons produced throughout exercise (Hochachka and
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Mommsen, 1983):
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ATPGLY = HCK++Hβ+-HOX++Hefflux+
[A4]
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HCK+ (in mM.min-1) was calculated from the changes in [PCr] and from the stoichiometric
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coefficient (γ):
HCK+ = -γ  ATPCK
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[A5]
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where γ is the proton stoichiometric coefficient of the coupled Lohmann reaction as described
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previously (Kushmerick, 1997).
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Hβ+ (in mM.min-1) was calculated from the apparent buffering capacity βtotal (in slykes,
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millimoles of acid added per unit change in pH) and from the rate of pH changes:
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[A6]
βtotal = βnon-bicarbonate-non-Pi + βPi + βPME + βbicarbonate,
[A7]
βnon-bicarbonate-non-Pi = βa - (βPi + βPME)
[A8]
Where
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Hβ+ = -βtotal  dpH/dt
where
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in which βa was determined from the initial change in [PCr] (Δ[PCr] ini) and alkalinization of pH
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(pHini) (2):
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βa = γ  Δ[PCr]ini/ΔpHini
[A9]
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βPi and βPME were determined based on the dissociation constant of the buffer (K) according to
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the standard formula (Conley et al., 1998):
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βx = (2.303  H+  K  [X])/(K + H+)2
[A10]
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where X is either Pi or PME and K = 1.77  10–7 for Pi and 6.3  10–7 for PME.
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In agreement with previous studies and assuming that muscle is a closed system during exercise
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(Conley et al., 1998, Kemp et al., 1993), βbicarbonate was set to zero.
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HOX+ (in mM.min-1) was calculated from the factor m = 0.16/[1 + 10(6.1 – pH)], which accounts for
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the amount of protons produced through oxidative ATP production:
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HOX+ = m  Q
[A11]
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Hefflux+ (in mM.min-1) was calculated for each time point of exercise using the proportionality
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constant λ relating proton efflux rate to ΔpHexo:
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Hefflux+ = ΔpHexo
[A12]
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This proportionality constant λ (in mM.min-1.pH.unit-1) was calculated during the recovery
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period:
λ = -Veff/pH
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[A13]
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During this period, PCr is regenerated throughout the CK reaction as the consequence of
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oxidative ATP production in mitochondria. Thus, Hefflux+ can be calculated from the rates of
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proton production from the CK reaction (HCK+, in mM.min-1) and mitochondrial ATP
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production (HOX+, in mM.min-1) on one side and the rate of pH changes on the other side. At this
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time ATP production is exclusively aerobic, and lactate production is considered as negligible:
Veff = βtotal  dpH/dt + γ  Vrecov PCr + m  Q
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[A14]
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Total ATPase Rate
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The total ATPase rate (ATPTOT, in mM.min-1) was calculated for each time point as:
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ATPTOT = ATPOX + ATPCK + ATPGLY
[A15]
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Energy cost of contraction (in mM.min-1.W-1) was also calculated as the ratio between ATPTOT
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and power output (W). Aerobic ATP cost of contraction (ATPAER, in mM.min-1.W-1) was
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calculated throughout the whole exercise period as the ratio between the mean amount of
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ATPOX produced during the exercise and mean power output produced during the same period
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of time. Anaerobic ATP cost of contraction was calculated similarly from ATP production by
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CK reaction and glycolysis (ATPCK + ATPGLY).