The main function of glycolysis and oxidative phosphorylation is to produce cellular energy in the form of ATP. In the present paper we propose a link between both of these energy-regulatory processes in the form of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CytOx (cytochrome c oxidase). GAPDH is the sixth enzyme of glycolysis, whereas CytOx is the fourth complex of the mitochondrial oxidative phosphorylation system. In MS analysis, GAPDH was found to be associated with a BN-PAGE (blue native PAGE)-isolated complex of CytOx from bovine heart tissue homogenates. Both GAPDH and CytOx are highly regulated under normal energy metabolic conditions, but both of these enzymes are highly deregulated in the presence of oxidative stress. The interaction of GAPDH with CytOx could be the point of interest as it has already been shown that GAPDH protein damage results in a marked decrease in cellular ATP levels. On the other hand, decreasing the ATP/ADP ratio may ultimately result in switching off the allosteric ATP inhibition of CytOx leading to increased ROS (reactive oxygen species), cytochrome c release and apoptosis. Moreover, we have previously reported that allosteric ATP inhibition of CytOx is responsible for keeping the membrane potential at low healthy values, thus avoiding the production of ROS and this allosteric ATP inhibition is switched on at a high ATP/ADP ratio. So, in the present paper, we propose a scheme that could prove to be a link between these two enzymes and their role in the prevalence of diseases.
In eukaryotic cells, most of the energy is produced in the form of ATP through the processes of glycolysis (in the cytosol) and oxidative phosphorylation (in mitochondria). The sixth step of glycolysis is catalysed by GAPDH (glyceraldehyde-3-phosphate dehydrogenase), which converts glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate in the presence of NAD+ and Pi . In addition to cytoplasm, GAPDH is also found in particulate fractions in mitochondria  and, because of its multiple roles in various cellular processes including oxidative phosphorylation, GAPDH is said to be more than a ‘housekeeping protein’ .
The oxidative phosphorylation system consists of five complexes (I–V) located at the inner membrane of mitochondria. CytOx (cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain responsible for converting molecular oxygen into water along with the translocation of protons across the mitochondrial inner membrane. This enzyme is said to be mainly regulated by allosteric effectors, subunit isoform expression and phosphorylation/dephosphorylation .
The proposed main role of CytOx is to regulate mitochondrial respiration by allosteric ATP inhibition in a way that keeps the mitochondrial membrane potential at low healthy values, thus avoiding the production of ROS (reactive oxygen species) . The deregulation of this enzyme through switching off the allosteric ATP inhibition leads to the increased production of ROS, which are responsible for many diseases including myocardial infarction [6,7]. On the other hand, GAPDH also has a metabolic switch function in the antioxidant response: it is inhibited by the oxidative stress condition [8,9], which makes it one of the most sensitive indices to myocardial hypoxaemia . Moreover, it has also been shown that the expression of the GAPDH gene displays high expression variability in mouse myocardial infarction tissues .
On the basis of the interesting fact that both GAPDH and CytOx are highly influenced by oxidative stress conditions (increased ROS production) in turn leading to various disease conditions, it gives an idea of their possible interaction in the energy metabolic processes. But the question remains, does this interaction exist in normal physiological conditions and, if so, does it have any effect on the respiration and kinetics of intact mitochondria? Moreover, how does this association of GAPDH with CytOx ultimately change the kinetics of the enzyme, especially the allosteric ATP inhibition of CytOx (the proposed second mechanism of respiratory control).
Physiological and pathophysiological roles of GAPDH
GAPDH is a soluble protein and participates in many physiological and pathophysiological processes. Other than its role in glycolysis, various non-glycolytic roles of GAPDH have also been described [1,12]. These diverse functions of GAPDH are likely to be dependent on its post-translational modifications, oligomerization and mainly its subcellular localizations (including cytoplasm, vesicles, nucleus and mitochondria) . The phosphorylation status of GAPDH regulates both its glycolytic and non-glycolytic functions . In the case of its pathophysiological role, it has been shown that pro-apoptotic mitochondrial membrane permeabilization is induced by GAPDH translocation to mitochondria. Exogenous expression of GAPDH in the mitochondria causes loss of the inner transmembrane potential, matrix swelling and thus permeabilization of the inner mitochondrial membrane 
On the other hand, Yao et al.  described an anti-apoptotic role of GAPDH in phenylephrine-induced cardiomyocyte protection by an increase in GAPDH glycolysis activity that mainly affects the mitochondria-induced apoptosis by depolarization of mitochondrial membrane potential. Another study reports that GAPDH protects HeLa cells from death by providing enough ATP to maintain the mitochondrial membrane potential . Similarly, GAPDH protein damage resulted in marked depletion of cellular ATP levels . Furthermore, it has also been shown that treatment of cardiomyocytes with iodoacetate inactivates GAPDH, resulting in almost 50% of the maximal ATP loss following H2O2 overload . Moreover, higher GAPDH activity is correlated with less apoptosis of cardiac muscle cells in ischaemia/reperfusion of isolated rat hearts . However, in another study, GAPDH inhibition and glutathionylation has been observed in cardiac tissue upon ischaemia/reperfusion . Iodoacetic acid has been shown to preferentially inhibit the GAPDH activity  that results in an increased cardiomyocyte apoptosis. In a rat model of cardiac ischaemia, the effect of GAPDH overexpression was examined and it has been shown that GAPDH overexpression and activation caused a reduction in cardiomyocyte apoptosis and myocardial infarct size . Inhibition of GAPDH activity due to oxidative stress leads to many other diseases such as Alzheimer's disease  and hyperglycaemia in diabetes [21–24].
Possible interaction of GAPDH and CytOx
By immunogold labelling and transmission electron microscopy of ‘normal’ rat liver tissues, it has been shown that GAPDH not only is localized in the cytosol, nucleus, plasma membrane and endoplasmic reticulum, but also was detected, unambiguously, in the mitochondria localized at the proximity of the mitochondrial membrane and at a relative proportion of 24% (analysed by immunoblot densitometry). Furthermore, it has been described that the levels of GAPDH in the mitochondria are low under basal conditions, but they are elevated under stressed conditions, such as serum deprivation and exposure to DNA-damaging agents . Most interestingly, the potential lethal role of GAPDH in mitochondria has always been proposed, but the function of GAPDH in mitochondria under normal physiological conditions has never been stated.
In our experimental settings, we have repeatedly found GAPDH together with CytOx when the enzyme was isolated by BN-PAGE (blue native PAGE) from bovine heart tissue homogenate (R. Ramzan, U. Linne and S. Vogt, unpublished work). These results were confirmed by MS and protein identification was performed using different databases. Determining whether this presence of GAPDH together with CytOx has an influence on its activity was the next step. The kinetic measurements of CytOx activity by polarography give a better understanding of complex IV activity. According to the first description by Kadenbach and Arnold , the second mechanism of respiratory control is based on the ATP-dependent allosteric inhibition of CytOx, which is switched on under high ATP/ADP ratios. We found a similar inhibitory effect on the kinetics of CytOx activity when GAPDH+GAP+NAD+ was used instead of ATP+PEP (phosphoenolpyruvate)+PK (pyruvate kinase) (Figure 1). Hence the close relationship between GAPDH activity and CytOx function is the focus of our future work that could show the direct influence of substrate level phosphorylation upon oxidative phosphorylation. Figure 2 presents the proposed scheme that shows the interaction of GAPDH with CytOx. Generally, GAPDH is a part of the Embden–Meyerhof–Parnas pathway performing glycolysis followed by phosphoglycerate kinase and PK reactions and thus producing two molecules of ATP. Therefore, in order to support oxidative phosphorylation, the ATP pool is kept at high levels through substrate level phosphorylation that may result in ATP-dependent inhibition of CytOx .
Polarographic measurements of CytOx activity in rat heart mitochondria at increasing concentrations of cytochrome c
Proposed scheme for the possible interaction of GAPDH and CytOx
In the past, it has always been stated that GAPDH is a part of glycolysis, but years of research have shown that, other than its glycolytic role, it is also involved in extra-glycolytic functions under both physiological and pathophysiological conditions. Moreover, under basal conditions, GAPDH has been shown to be localized also in mitochondria as has been shown in normal rat liver tissue. But the role of GAPDH in mitochondria under normal physiological conditions has never been stated.
Interaction of GAPDH together with CytOx could explain their possible roles in regulating the normal energy processes. Most importantly, it could also explain the first descibed cytosolic signal influencing the second mechanism of respiratory control that is based on the high ATP/ADP ratios inhibiting CytOx activity in the mitochondria. GAPDH/GAP/NAD+ influence the kinetics of CytOx activity in exactly the same manner as that of ATP/PEP/PK. Moreover, both GAPDH and CytOx are highly regulated under normal energy metabolic conditions, but both of these enzymes are highly deregulated in the presence of oxidative stress. It has been shown that GAPDH protein damage results in a marked decrease in the cellular ATP level that on the other hand can result in a decrease in the ATP/ADP ratio that may ultimately switch off the allosteric ATP inhibition of CytOx leading to increased ROS production, cytochrome c release and apoptosis.
Bioenergetics in Mitochondria, Bacteria and Chloroplasts: Third Joint German/UK Bioenergetics Conference, a Biochemical Society Focused Meeting held at Schloss Rauischholzhausen, Ebsdorfergrund, Germany, 10–13 April 2013. Organized and Edited by Fraser MacMillan (University of East Anglia, Norwich, U.K.) and Thomas Meier (Max Planck Institute of Biophysics, Frankfurt am Main, Germany).
We thank Professor Bernhard Kadenbach for his helpful discussions and suggestions.
These authors contributed equally to this paper.