Estimation of flux control coefficients from inhibitor titrations by non-linear regression

AbstractA mathematical model was developed to estimate flux control coefficients (Co) from titration studies with specific non-competitive inhibitors. In contrast to the normally used graphical determination the model pays regard to the dissociation equilibrium (kD) that exists between inhibitor and its binding sites (Eo) as well as to an objective estimation of the initial slope. The model was used for the analysis of titration experiments where the respiration of rat liver mitochondria was inhibited with carboxyatractyloside and antimycin A. It is shown that the graphical estimation of Eo and Co lead to significant overestimation if the ratio Kd/Eo is larger than 10−4 which can be avoided by using our model

Extramitochondrial Ca2+ in the Nanomolar Range Regulates Glutamate-Dependent Oxidative Phosphorylation on Demand

We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca2+ in physiological concentration ranges (S0.5 = 360 nM Ca2+). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca2+ uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca2+-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca2+. At micromolar concentrations, Ca2+ can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca2+ regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca2+

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

AbstractCarboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 μM CAT and 75 μM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 μM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 μM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP.We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression

Cytosolic Ca^{2+} regulates the energization of isolated brain mitochondria by formation of pyruvate through the malate-aspartate shuttle

The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca2+cyt (cytosolic Ca2+) (S0.5=225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca2+cyt (S0.5=60±10 nM). At higher Ca2+cyt (&amp;gt;600 nM), BM accumulate Ca2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca2+-induced increments of state 3 respiration decrease with substrate in the order glutamate&amp;gt;α-oxoglutarate&amp;gt;isocitrate&amp;gt;α-glycerophosphate&amp;gt;pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca2+cyt, we show that the formation of pyruvate is tightly controlled by Ca2+cyt. Through its common substrate couple NADH/NAD+, the formation of pyruvate by LDH (lactate dehydrogenase) is linked to the MAS (malate–aspartate shuttle) with aralar as a central component. A rise in Ca2+cyt in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to 5-fold enhancement of OXPHOS (oxidative phosphorylation) rates that is due to an increased substrate supply, acting in a manner similar to a ‘gas pedal’. In contrast, Ca2+mit (intramitochondrial Ca2+) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca2+cyt is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons.</jats:p

Exclusive activation of glutamate-dependent state 3 respiration of brain mitochondria by extramitochondrial Ca²⁺ in the nanomolar range.

<p>(A,E) Respirograms of rat brain mitochondria were obtained by high-resolution respirometry. (A) Isolated rat brain mitochondria were incubated in EGTA medium (Ca²⁺_free = 0.15 µM) in the presence of 10 mM glutamate and 2 mM malate as substrates. Additions: M, 0.06 mg/ml brain mitochondria, A, 2.5 mM ADP to activate the phosphorylation-related respiration (state 3); Ca²⁺_4,9, 4.9 µM Ca²⁺_free; S, 10 mM succinate as substrate of respiratory chain complex II; C, 5 µM carboxyatractyloside to block the adenine nucleotide translocase. Blue lines indicate the oxygen concentration and red lines represent respiration rates (nmol O₂/mg mitochondrial protein/min). (B) Means of state 3 respiration±S.E. as measured in experiments shown in A without (black columns, n = 6) or with 250 nM RR, an inhibitor of mitochondrial Ca²⁺ uptake (red columns, n = 6). First group of columns, state 3 at Ca²⁺_free = 0.15 µM. Second group, state 3 with Ca²⁺_free = 4.9 µM. Third group, state 3 with Ca²⁺_free = 4.9 µM in the additional presence of 10 µM succinate. *, p<0.05. (C) As B, but derived from experiments with 10 mM pyruvate + 2 mM malate as substrates. *, p<0.05. (D) As B, but derived from experiments with 10 mM succinate + 2 µM rotenone as substrate. (E) Ca²⁺ titration of state 3_glu/mal by stepwise increase of Ca²⁺ as indicated either without (E,F) or with (F) 250 nM RR. (F) Incremental accretions of Ca²⁺-induced state 3_glu/mal were plotted against the fluorimetrically measured Ca²⁺ activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008181#pone-0008181-g001" target="_blank">Fig. 1F</a>), allowing the calculation of the half-activation constant (S_0.5) and the maximum velocity (V_max) using the SigmaPlot kinetic module as given in the text. (G) Rates of state 3_glu/mal respiration obtained by Ca²⁺ titrations under various conditions. (○) Control mitochondria were investigated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008181#pone-0008181-g001" target="_blank">Fig. 1E</a>. (□) As (○), but in the additional presence of 10% dextran 20. (▿) As (○), but in the additional presence of 1 mM CsA. (▵) as (○), but mitochondria isolated without digitonin were used. (◊) as (○), but mitoplasts were used. () as (○), but mitochondria were uncoupled by 50 nM FCCP from the beginning of experiments, and then Ca²⁺ titration was performed. (▴) as (○), but Ca²⁺ was adjusted at the beginning of experiments as indicated. Thereafter, 100 µM ADP was added, causing short transitions between the active and resting states of respiration. After reaching state 4 respiration, FCCP titrations were performed to uncouple respiration and ATP generation. Maximum respiration rates were obtained at 60 or 80 nM FCCP and were plotted against the Ca²⁺_free value for the respective incubation. Data are means±S.E. of 4 independent experiments.</p