MALATE-ASPARTATE SHUTTLE AND EXOGENOUS NADH/CYTOCHROME C ELECTRON TRANSPORT PATHWAY AS TWO INDEPENDENT CYTOSOLIC REDUCING EQUIVALENT TRANSFER SYSTEMS

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α- ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate amino transferase

Inhibition by butylmalonate of proton influx in nonphosphorylating mitochondria

The impermeability of the inner membrane to protons is one of the four postulates of the chemiosmotic theory on the coupling mechanism between respiration and phosphorylation in mitochondria. However, oxygen uptake in isolated nonphosphorylating mitochondria requires that protons translocated from inside to outside must be, at least in part, retaken up. The nonohmic relationship between the respiration rate and the protonmotive force has been mainly ascribed to an increase in the proton conductance of the inner membrane (proton leak). In liver mitochondria oxygen pulse experiments the rate of both the efflux and the reentry of protons, linked to the oxygen consumption supported by succinate oxidation, is greatly stimulated by low concentrations of butylmalonate. The steady-state level of protons exported outside in the acidification-alkalinization cycle of the medium, generated by an oxygen pulse, is also increased but the rate of oxygen uptake is unaffected. However, in valinomycin-stimulated respiration butylmalonate inhibits the ratio of proton influx/oxygen consumption by 50% and also stimulates the ratio of proton efflux/oxygen consumption by 50%. Titration of the butylmalonate effect gives a saturation curve with a half-maximal effect at 5 microM. Identical results are obtained inthe presence of oligomycin which excludes the involvement of the ATP-synthase complex. The data obtained are not in contrast with the existence in the inner membrane of a channel-like system inhibited by butylmalonate and involved, together with other systems, in promoting the backflow of protons in nonphosphorylating state 4 respiration. Such a system, similar to thermogenin, could be involved in tissues, other than adipose, in a more general thermogenesis program by promoting the dissipation as heat of the energy given by the electrochemical proton gradient. The possibility that butylmalonate might inhibit the proton movement associated with cation and anion transport in mitochondria has also been considered

Modulation of cytochrome c-mediated extramitochondrial NADH oxidation by contact site density

Data presented in previous reports suggest that in rat liver mitochondria a “bi-trans-membrane” electron transport pathway is present which promotes the transfer of reducing equivalents directly from cytosolic NADH to molecular oxygen inside the mitochondria. Here we show that the oxidation of external NADH is stimulated by atractylate 1 ADP and greatly inhibited by glycerol. These two conditions have been documented to promote the increase and the decrease respectively of the frequency of “contact sites” between the two mitochondrial membranes. NADH oxidation is not affected at all by glycerol and atractylate 1 ADP when TMPD and endogenous cytochrome c are utilized as electron carriers. The results obtained are consistent with the proposal that the bi-transmembrane electron transport chain might be localized at the level of respiratory contact sites having the function of promoting the oxidation of the surplus amount of cytosolic NADH. This electron transport pathway has been suggested to play a decisive role in the early stages of apoptosi

Valinomycin induced energy-dependent mitochondrial swelling, cytochrome c release, cytosolic NADH/cytochrome c oxidation and apoptosis

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that ‘‘respiratory contact sites’’ are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program