Identification and characterization of a protozoan uncoupling protein in Acanthamoeba castellanii.

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the divergence points of plants, animals, and fungi. The existence of UCP in A. castellanii (AcUCP) has been revealed using antibodies raised against plant UCP. Its molecular mass (32,000 Da) was similar to those of plant and mammalian UCPs. The activity of AcUCP has been investigated in mitochondria depleted of free fatty acids. Additions of linoleic acid stimulated state 4 respiration and decreased transmembrane electrical potential (DeltaPsi) in a manner expected from fatty acid cycling-linked H(+) reuptake. The half-maximal stimulation by linoleic acid was reached at 8.1 +/- 0.4 microM. Bovine serum albumin (fatty acid-free), which adsorbs linoleic acid, reversed the respiratory stimulation and correspondingly restored DeltaPsi. AcUCP was only weakly inhibited by purine nucleotides like UCP in plants. A single force-flow relationship has been observed for state 4 respiration with increasing concentration of linoleic acid or of an uncoupler and for state 3 respiration with increasing concentration of oligomycin, indicating that linoleic acid has a pure protonophoric effect. The activity of AcUCP in state 3 has been evidenced by ADP/oxygen atom determination. The discovery of AcUCP indicates that UCPs emerged, as specialized proteins for H(+) cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world

Evidence for cooperative effects in the exchange reaction catalysed by the oxoglutarate translocator of rat-heart mitochondria.

The initial rates of the exchange external oxoglutarate/internal malate through the inner membrane of rat-heart mitochondria, for various concentrations of the two substrates, have been reinvestigated for an extended range of concentrations of the external oxoglutarate. This has been made possible by use of the inhibitor-stop technique that allows 100 times smaller incubation times than the centrifugation-stop technique used previously. Under the experimental conditions the uptake of the external-labelled oxoglutarate into the mitochondrial-matrix space is mediated by the oxoglutarate translocator performing a ono-to-one exchange of the anions oxoglutarate (external) and malate (internal). Two intermediary-plateau regions are observed in the kinetic saturation curve of the translocator by the external oxoglutarate, revealing a complex rate equation which is found to be the product of two one-substrate functions. Analysing these features it is shown that the model, proposed earlier, of a "double carrier" as catalyst in a rapid-equilibrium random bi-bi mechanism, is still applicable but that several external binding sites have to be considered. As already noticed the external and the internal substrates bind to their respective sites independently of each other. Furthermore, some additional requirements imposed by the observed kinetics suggest that the exchange reaction is performed by only one translocator species made of identical interacting subunits. The anion exchange is tentatively viewed as a rotation of a subunit around an axis situated in the plane of the membrane after two independent local configuration changes induced by the binding of the two substrates on this subunit

Respiratory chain network in mitochondria of Candida parapsilosis: ADP/O appraisal of the multiple electron pathways.

In this study we demonstrated that mitochondria of Candida parapsilosis contain a constitutive ubiquinol alternative oxidase (AOX) in addition to a classical respiratory chain (CRC) and a parallel respiratory chain (PAR) both terminating by two different cytochrome c oxidases. The C. parapsilosis AOX is characterized by a fungi-type regulation by GMP (as a stimulator) and linoleic acid (as an inhibitor). Inhibitor screening of the respiratory network by the ADP/O ratio and state 3 respiration determinations showed that (i) oxygen can be reduced by the three terminal oxidases through four paths implying one bypass between CRC and PAR and (ii) the sum of CRC, AOX and PAR capacities is higher than the overall respiration (no additivity) and that their engagement could be progressive according to the redox state of ubiquinone, i.e. first cytochrome pathway, then AOX and finally PAR