Analysis of the Reactivity of SH-Reagents with the Mitochondrial Phosphate Carrier (1974)

The inhibition of mitochondrial Pi transport by SH-reagents was investigated together with incorporation of SH-reagents into mitochondrial membranes. The reactivity of the Pi carrier SH-group is influenced by several factors during the preincubation with the SH-reagent. By using more or less permeant maleimide derivatives, ethylmaleimide and N-(N-acetyl-4-sulfamoyl-phenyl) maleimide (ASPM), different reactivities can be attributed to accessibility variations. Differences in reactivity become apparent only on short-term incubations with SH-reagents, which allow measurements of reaction rates. The following results were obtained: 1 The reaction rate of ASPM with the Pi carrier is about three-times slower than that of ethylmaleimide. 2 With increasing pH the reactivity of the Pi carrier decreases with ASPM but increases with ethylmaleimide. 3 Pi protects partially against inhibition both by ethylmaleimide and ASPM with a Km between 2 and 10 mM, slightly competitive with the SH-reagent. For protection Pi must be allowed to be taken up by the mitochondria indicating that endogenous Pi protects better than exogenous Pi. 4 The incorporation of ethylmaleimide and ASPM into mitochondria shows smaller differences than inhibition of Pi transport. On titration with SH-reagents the inhibitor of the Pi carrier begins when 11 out of a total of 20 μmol SH-groups/g protein have been reacted. Inhibition is then complete when 15 μmol/g protein have been titrated. Thus 2–4 μmol SH-reagent cause the Pi carrier to pass from an active to an inactive form. This number probably includes more SH-groups than can be attributed to the Pi carrier. 5 The SH-reactivity of the Pi carrier is strongly influenced by uncouplers, ionophores and respiratory inhibitors. The strongest increase of reactivity is obtained with valinomycin, less with uncoupler and none with nigericin. The protective effect of Pi is then completely abolished. On the other hand, valinomycin plus uncoupler does not increase the sensitivity to SH-reagents. 6 Succinate and other respiratory substrates decrease reactivity and increase protection by Pi, whereas malonate increases inhibition. This shows that the removal of endogenous Pi by exchange with a nonrespiring dicarboxylate reverses the protective effect of Pi. The variability of SH-reactivity is interpreted in terms of accessibility of ASPM. The protection of the Pi carrier is mainly based on reorientation of the carrier inside and outside the membrane. A carrier model is derived from the data in which an equilibrium exists between a protonized immobile form (CH+) of the carrier and a dissociated mobile form (C). Only the CH+ form can bind with mono-anionic Pi− and gives the mobile Pi-carrier complex. Thus the Pi-carrier complex will distribute with ΔpH across the membrane opposite to Pi. The protection by Pi is mainly due to endogenous Pi moving Pi-carrier complex inside, the activation by H+ mainly due to moving empty carrier outside. The model is in accordance with an electroneutral Pi transport. It explains the majority of data, in particular the stimulation by H+ and by valinomycin and also the preferential protection by endogenous Pi.