Addition of triiodothyronine at 10 pM in vitro to hypothyroid rat liver mitochondria doubles the rate of the adenine nucleotide transporter at low ADP concentrations. Nicotinamide abolishes this effect in parallel with its inhibition of the ADP-ribosylation of an inner membrane protein identical in size to the transporter. Nieotinamide also renders euthyroid preparations indistinguishable from hypothyroid ones. A mechanism is offered to explain these findings in which it is proposed that the adenine nucleotide transporter is a true allosteric protein and that its covalent modification by ADP-ribosylation increases the stability of the less favoured externally-facing C-conformation and thus increases the proportion of transporters in this orientation: although the C-conformation is significantly more leaky to cations than the tight matrix-facing M-conformation, this enhances ADP import. This model is shown to offer an explanation not only for the transport effects of T3 but also for those of oxidative stress and ADP-ribosylation inhibitors on Ca 2+, H ÷ and K + transfer across the mitochondrial inner membrane. Ca 2+ at 30 nM appears to stabilize the Mconformation of the transporter by a mechanism other than ADP-ribosylation.

It is well established that reoxygenation of myocardial tissue after a period of hypoxia or ischaemia results in an increase in intracellular calcium [ 11 and the release of cytosolic contents as a consequence of cell lysis 121. As yet the mechanism and sequence of events involved in the so-called 'oxygen paradox' are unresolved. An insight into the mechanism of reoxygenation damage may be gained by investigating those cells which have not sustained lethal damage.Under normal circumstances the energy demands of active cardiac tissue are almost entirely satisfied by mitochondrial ATP synthesis. Hence the effect of myocardial reoxygenation o n mitochondrial function is of the greatest significance. Consequently, the effects of hypoxia and/or reoxygenation o n mitochondrial function has been investigated.Isolated rat hearts were subjected to retrograde perfusion by the Langendorff method with Krebs bicarbonate buffer. After an initial 5 min equilibration period of perfusion with 1 1 m~-glucose and gassing with 02/COz ( 19 : 1 ), the control, normoxic hearts were then gassed for a further 45 min with glucose-free buffer. Hypoxic hearts were those gassed with N2/C0, (10: 1) for 40 min with glucose-free buffer, while reoxygenated hearts were subject to a further 5 min of perfusion with O,/CO, (1Y:l). At the end of the perfusion, mitochondria were prepared by trypsin digestion. homogenization and differential centrifugation.The activity of the electron transport chain was determined by using a Clark-type electrode, the mitochondrial respiration rates being measured in the presence of both NAD-linked (pyruvate + malate) and FAD-linked (succinate) substrates. The activities of the individual respiratory chain complexes isolated from the membrane by freeze/thawing were determined by assaying NADH-cytochrome c reductase activity (complex 1-111) and succinate-cytochrome c reductase activity (complex 11-111). Table 1 shows that there is a 44% decrease in the state 3 respiration rate with NAD-linked substrates, but no reduction in the succinate-linked rate, in mitochondria isolated from hearts subjected to hypoxia and reoxygenation, but not hypoxia alone. The respiratory control ratios are reduced after reoxygenation, but without an increase in state 4 respiration (data not shown), which suggests that membrane integrity is unaffected. The reduction in complex I activity is confirmed by the enzyme assay data in Table 1. Reoxygenated isolated hypoxic heart mitochondria do not show the complex I defect, suggesting that the mechanism involves the intervention of the whole myocyte or tissue. Isolated adult cardiomyocytes subject to hypoxia and reoxygenation have been shown not to undergo cell lysis 131, but preliminary studies have shown that they do exhibit the complex I defect. This suggests that the whole tissue is not necessary for the defect t o be expressed, and that the relationship of the mitochondrial defect and cell lysis may be indirect.Evidence has been presented that the mitochondrial calcium uptake inhibitor Ruthenium...

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By using an assay based on the precipitation of intact 14C-labelled substrate, an activity has been located in the mitochondrial fraction of rat liver which selectively hydrolyses the 3′ ester link in the fairly recently discovered oligomeric tetraphosphate derivative of ATP and glyceric acid for which the structure 3-phospho[glyceroyl-gamma-triphospho-5′-adenosine-3′-3-phospho]n-glyceroyl- gamma-triphospho-5′-adenosine has been proposed [Hutchinson, Morris & Mowbray (1986) Biochem. J. 234, 623-627]. This enzyme activity (Mr 85,000) has been purified approx. 30-fold from washed mitochondria by (NH4)2SO4 precipitation and f.p.l.c. The apparent Km for substrate (adenosine equivalents) is around 35 microM. The recovery of total activity is about 20%, and this, allied to the relatively low Vmax. found in contrast with the rapid turnover of oligomer seen in post-ischaemic tissues, suggests that some activating factors have been lost in purification. Percoll-gradient studies confirm that the activity is mitochondrial and not lysosomal or endoplasmic-reticular. The activity is latent in intact mitochondria; it is not, however, associated with intact inner-membrane vesicles but released during their preparation, implying an intermembrane-space location. The product of the enzyme is proposed to be the monomeric unit 3-phosphoglyceroyl-gamma-triphospho-5′-adenosine, from which digestion with snake-venom phosphodiesterase releases ADP.