2 research outputs found
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
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