23 research outputs found
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
NADH/CYTOCHROME C ELECTRON TRANSPORT SYSTEM AND MITOCHONDRIAL PERMEABILITY TRANSITION AS TWO DISTINCT AND INDEPENDENT PROCESSES
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
In Highly Hypotonic Mitochondria The External Membrane Does Not Burst But Maintains Its Protective Envelope Function
Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system
Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxidation
of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto-c electron
transport pathway. The data presented in this article show that the protective
effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes
a differential effect on both the additional release of endogenous cyto-c and on the increased rate of NADH oxidation, depending on
whether it is added before or after the mitochondria. At the same time,magnesium prevents or almost completely removes the binding of exogenously added cyto-c. We suggest that, in physiological low-amplitude swelling, magnesium ions may have the function, together with other factors, of
modulating the amount of cyto-c molecules transferred from the mitochondrial
intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto-c electron transport pathway
Glycolytic enzyme upregulation and numbness of mitochondrial activity characterize the early phase of apoptosis in cerebellar granule cells
Alzheimer's disease (AD) and cancer proceed via one or more common molecular mechanisms: a metabolic shift from oxidative phosphorylation to glycolysis-corresponding to the activation of the Warburg effect-occurs in both diseases. The findings reported in this paper demonstrate that, in the early phase of apoptosis, glucose metabolism is enhanced, i.e. key proteins which internalize and metabolize glucose-glucose transporter, hexokinase and phosphofructokinase-are up-regulated, in concomitance with a parallel decrease in oxygen consumption by mitochondria and increase of L-lactate accumulation. Reversal of the glycolytic phenotype occurs in the presence of dichloroacetate, inhibitor of the pyruvate dehydrogenase kinase enzyme, which speeds up apoptosis of cerebellar granule cells, reawakening mitochondria and then modulating glycolytic enzymes. Loss of the adaptive advantage afforded by aerobic glycolysis, which occurs in the late phase of apoptosis, exacerbates the pathological processes underlying neurodegeneration, leading inevitably the cell to death. In conclusion, the data propose that both aerobic, i.e. Warburg effect, essentially due to the protective numbness of mitochondria, and anaerobic glycolysis, rather due to the mitochondrial impairment, characterize the entire time frame of apoptosis, from the early to the late phase, which mimics the development of AD
Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain
Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity
Proton translocation linked to the activity of the bi-trans-membrane electron transport chain
Recently we have proposed and presented evidence suggesting the existence of a "bi-trans-membrane" electron transport chain, located at the contact sites between outer and inner mitochondrial membranes, which can be utilized to promote either the oxidation of exogenous NADH in the presence of catalytic amounts of added cytochrome c or the reduction of exogenous cytochrome c supported by the oxidation of respiratory substrates present inside the mitochondria. Here we show that the oxidation of exogenous NADH is accompanied by a net alkalinization of the incubation medium preceded by a transient acidification phase. In oxygen-pulse experiments, the alcohol oxidation (induced by the addition of alcohol dehydrogenase) was used to mimic a cytosolic source of reducing equivalents. Oxygen pulses promote an acidification-alkalinization proton cycle which is insensitive to antimycin and myxothiazol inhibitory effect, is stimulated by valinomycin, inhibited by trypsin-aprotinin complex, abolished by the protonophore carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and is absent or at least inverted (alkalinization-acidification cycle) in broken mitochondria. The oxidation of cytosolic substrates, mediated by the bi-trans-membrane electron transport chain, does not involve endogenous cytochrome c and is associated with a vectorial proton translocation from the inside to the outside of the mitochondria. In the out-->in electron transport pathway the components involved appear to be cytosolic reduced substrates-->NADH produced by cytosolic dehydrogenases activity-->NADH-cytochrome b5 oxidoreductase complex leaning out the external side of the external membrane-->exogenous cytochrome c-->cytochrome oxidase of contact sites-->molecular oxygen. The possible components of the in-->out pathway are matrix respiratory substrates-->primary dehydrogenases of the matrix-->Complexes I, II, and III of the respiratory chain present in the inner membrane-->NADH-cytochrome b5 oxidoreductase system of the external membrane-->exogenous cytochrome c-->additional cytosolic electron acceptors or, alternatively, cytochrome oxidase of contact sites. The two pathways can be considered a bi-trans-membrane electron channeling system which, at the level of bridges set up by the contact points between the outer and the inner mitochondrial membrane, may represent a link between the redox processes occurring inside with those present outside the mitochondrion