The kinetics of citrate uptake by malate-loaded mitochondria were measured using the inhibitor stop method and analysed for possible carrier mechanisms. 1.The citrate exchange is found to follow a first order reaction with a constant k of 1.18 min-l and the corresponding rate of citrate uptake of 13.4 pmolxmin-l x g protein-l (at 0.5 mM citrate and 9 "C). The half time is 34 see.2. The temperature dependence of citrate exchange was measured in the range between 0 and 14 "C. An activation energy of 20.1 kcal and a Q,, of3.6 can be calculated from the Arrhenius 3. The concentration dependence of citrate exchange reveals hyperbolic saturation characteristics. The K , and V values for the rate of citrate uptake are 0.12 f 0.01 mM and 22.5 & 1.8 pmol citrate x mir-l x g protein-l respectively at 9 "C in 11 experiments. 4.The rate of citrate uptake has a pH optimum of about 7. The inhibition by higher pH is competitive with citrate. At pH lower than 7, V is decreased, indicating that the citrate transporting system is inactivated by acid pH.5. The rate of citrate exchange is inhibited in a competitive manner by cis-aconitate, threo-D,-isocitrate, 1,2,3-propanetncarboxylate, 1,2,3-benzenetricarboxylate, citrate and propylcitrate. Other tricarboxylates, however, such as trans-aconitate and 1,3,5-pentanetricarboxylate have no effect on citrate exchange, even when added in large excess.6. Succinate, malonate, oxaloacetate and oxomalonate inhibit the rate of citrate uptake. In contrast, glutarate, adipate, pimelate, 2-oxoglutarate, aspartate and glutamate do not. Maleate, but not the trans isomer fumarate, also inhibits citrate uptake. The inhibition of citrate uptake is great,er with malate than with succinate.7. The rate of citrate uptake is also inhibited by the nonpenetrant anions phenylsuccinate, butylmalonate, benzylmalonate and pentylmalonate, previously thought to be specsc inhibitors of the dicarboxylate carrier.8. The inhibition of the rate of citrate uptake by dicarboxylates and their analogues is found to be competitive. The affinity for dicarboxylates is lower than for tricarboxylates (Ki is 0.7 mM for malate).9. Phosphoenolpyruvate strongly inhibits the rate of citrate uptake, while Pi and pyruvate have only a slight effect. The inhibition by phosphoenolpyruvate is shown to be competitive (Ki is 0.11 mM). 10.It is concluded that the tricarboxylate carrier has a single binding site for tricarboxylates, plot.phosphoenolpyruvate and dicarboxylates. The implications of these findings are discussed.Several pieces of evidence indicate that the transport of tricarboxylates through the mitochondria1 membrane is mediated by a specific carrier, which catalyzes an exchange diffusion of a tricarboxylate for either a tricarboxylate or a dicarboxylate [1,2].To gain further insight into the catalytic mechanism and for a quantitative understanding of the physiological importance of the ticarboxylate carrier, direct kinetic studies are necessary.In this paper, quantitative values of the rate and related kinetic parameters of ...

The rate of uptake in mitochondria as catalyzed by the dicarboxylate carrier of dicarboxylates and, in the presence of N-ethylmaleimide, of inorganic phosphate, has been analyzed by using the inhibitor stop method.1. By comparing the dependence on substrate concentration of the rate of succinate, malonate and malate uptake, it is found that the K , (malate) = 0.23 < Km (malonate) = 0.37 < K m (succinate) = 1.17 mM. The V values, however, are not significantly different for all three dicarboxylates ( V = 70 pmoles/g protein x min at 9 "C).2. Succinate, malate and malonate are shown to be competitive with each other in the kinetics of uptake.3. The inhibition of the rate of malate uptake by 2-phenylsuccinate is found to be competitive. 4. Several "permeant" anionic substrates such as acetate, pyruvate, 3-hydroxybutyrate, glutamate and aspartate have no effect on the rate of dicarboxylate uptake, even when added in large excess.5. The rate of malonate uptake is inhibited by 2-oxoglutarate but unaffected by citrate, while the rates of succinate and malate uptake are decreased by both 2-oxoglutarate and citrate. Malate is inhibited more than succinate. The inhibition of the rate of malate uptake by citrate and 2-oxoglutarate is shown to be non-competitive. This is explained due to a re-exchange of accumulated malate by citrate or 2-oxoglutarate.6. In the presence of N-ethylmaleimide, externally added Pi inhibits the rate of dicarboxylate uptake. The Pi inhibition of malate uptake is found to be mixed with a larger share of noncompetitive than competitive inhibition.7. The V of the rate of Pi uptake is approximately equal to that of malate uptake in the presence of N-ethylmaleimide. The Ki is, however, higher for Pi (Ki = KO = 1.95 mM) than for malate.8. It is concluded firstly that the dicarboxylate carrier specifically transports either a dicarboxylate or Pi (but not both together), and secondly that the carrier has two separate binding sites, one specific for Pi and the other specific for the dicarboxylates.Detailed kinetic studies on the mitochondria1 carrier systems for anionic substrates are available only for the adenine nucleotide translocation [l] although kinetic data are of great importance for an understanding of the carrier-catalyzed transport processes. The kinetics of succinate uptake were reported previously [2,3] and shown to reveal saturation kinetics and a high temperature and pH dependence. Furthermore, it was found that succinate uptake appears to follow a first order type of reaction [2]. More recently, also applying the "inhibitor stop method", the kinetics of the efflux of dicarboxylates from the mitochondria were measured, using various inhibitors [4].In this paper, the kinetics of the uptake of various substrates linked to the dicarboxylate carrier are reported. The specificity, and the competition with other anions and between dicarboxylates, are measured and analyzed for possible carrier mechanisms. A preliminary account of the present work has been presented [5]. MATERIALS AND METHODS Mat...

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Rat heart mitochondrial membranes exposed to the free radicals generating system tert-butylhydroperoxide/Cu 2+ undergo lipid peroxidation as evidenced by the accumulation of thyobarbituric acid reactive substances. Mitochondrial lipid peroxidation resulted in a marked loss of both cytochrome c oxidase activity and cardiolipin content. The alterations in the properties of cytochrome c oxidase were confined to a decrease in the maximal activity (V m x ) with no change in the affinity (K m ) with respect to the substrate cytochrome c. Various lipid soluble antioxidants could prevent the lipid peroxidation reaction and the associated loss of cytochrome c oxidase activity. External added cardiolipin but no other phospholipids, nor peroxidized cardiolipin was able to prevent the loss of cytochrome oxidase activity induced by lipid peroxidation. These results establish a close correlation between oxidative damage to cardiolipin and alterations in the cytochrome oxidase activity and may prove useful in probing molecular mechanism of free radicals induced peroxidative damage of mitochondria which has been proposed to contribute to aging and to chronic degenerative diseases.z 1998 Federation of European Biochemical Societies.

In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC.The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD+FMN and FMN-riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification.Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover.