Abstract
Measurements of water proton spin relaxation enhancements (ε) can be used to discriminate high-affinity binding of Mn 2+ or Gd 3+ to biological membranes, from low-affinity binding. In rat liver mitochondria, ε b values of approx. 11 are observed upon binding of Mn 2+ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn 2+ uptake by liver mitochondria results in the subsequent decay of ε ∗. Comparison of ε ∗ with the initial velocity of Mn 2+ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 °C and 40 °C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn 2+ to the divalent cation pump. Binding of Mn 2+ to blowfly flight muscle also results in substantial ε ∗, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in ε ∗ due to uptake occurs after Mn 2+ is bound. Lanthanide ions are also bound and transported by mitochondria. Addition of Gd 3+ to pigeon heart or rat liver mitochondria results in ε b ≈ 5–6, which decays with similar kinetics in both systems. The uptake velocity of Gd 3+ in rat liver mitochondria is about 1 6 the rate with which Mn 2+ is transported. Lanthanides also diminish ε ∗ due to the addition of Mn 2+, and greatly retard the Mn 2+ uptake kinetics. The presence of carbonylcyanide- p-trifluoromethoxyphenylhydrazone depresses ε ∗ upon addition of Mn 2+ or Gd 3+ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn 2+, but does not affect ε ∗ significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of “de-energization”. Measurements of ε ∗ at several NMR frequencies indicate a correlation time ( τ b) for carrier-bound Mn 2+ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 °C and 30 °C. The 13 Kcal/mole activation energy for τ b suggests that the 11 ns time constant at room temperature represents the movement of the Mn II-carrier complex. On the other hand, τ b is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn 2+ binds to a small arm of the carrier which moves independently of the main body of any protein. In addition to Mn(H 2O) 6 2+, other complexes of Mn 2+ may also be bound and transported by rat liver mitochondria. Only a small increase in ε ∗ occurs upon addition of MnHPO 4, yet this species is accumulated by the mitochondria. Consequently, the carrier does not recognize divalent metal ions on the basis of charge.
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