Amoeba mitochondria possess a respiratory chain with two quinol-oxidizing pathways: the cytochrome pathway and the cyanide-resistant alternative oxidase pathway. The ADP/O method, based on the non-phosphorylating property of alternative oxidase, was used to determine contributions of both pathways in overall state 3 respiration in the presence of GMP (an activator of the alternative oxidase in amoeba) and succinate as oxidizable substrate. This method involves pair measurements of ADP/O ratios plus and minus benzohydroxamate (an inhibitor of the alternative oxidase). The requirements of the method are listed and verified. When overall state 3 respiration was decreased by increasing concentrations of n-butyl malonate (a non-penetrating inhibitor of succinate uptake), the quinone reduction level declined. At the same time, the alternative pathway contribution decreased sharply and became negligible when quinone redox state was lower than 50%, whereas the cytochrome pathway contribution first increased and then passed through a maximum at a quinone redox state of 58% and sharply decreased at a lower level of quinone reduction. This study is the first attempt to examine the steady-state kinetics of the two quinol-oxidizing pathways when both are active and to describe electron partitioning between them when the steady-state rate of the quinone-reducing pathway is varied.The mitochondrial respiratory chain of the amoeba Acanthamoeba castellanii possesses, like that of plant mitochondria (1), the following: (i) a cyanide-and antimycin-resistant alternative oxidase (AOX) 1 in addition to the conventional cytochrome c oxidase (2, 3); (ii) the rotenone-insensitive external NADH dehydrogenase, located on the outer surface of the inner mitochondrial membrane (4, 5); and (iii) two internal NADH dehydrogenases, the rotenone-sensitive complex I and the non-electrogenic rotenone-insensitive dehydrogenase (6).As in mitochondria from most higher plants, many fungi, and protozoa (7), the alternative pathway of amoeba mitochondria branches from the main respiratory chain at the level of quinone (Q), and electron flux through AOX is not coupled to the generation of protonmotive force and ADP phosphorylation (2). The cyanide-resistant respiration of amoeba mitochondria is strongly stimulated by purine nucleoside 5Ј-monophosphates: AMP, GMP (with the highest efficiency), and IMP (2,8,9). A similar effect of purine mononucleotides on the activity of the alternative pathway was observed in other microorganisms: Euglena gracilis (10), Moniliella tomentosa (11), Neurospora crassa (12), Paramecium tetraurelia (13), and Hansenula anomala (14). In contrast, the alternative oxidase of higher plant mitochondria is stimulated by ␣-keto acids, like pyruvate (7, 15, 16), but not by purine mononucleotides, whereas the ␣-keto acids do not stimulate the alternative respiration in amoeba mitochondria (17). These properties emphasize an important difference at the level of allosteric regulation between both types of AOX. However, monoclonal antibodie...

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Since yeast Saccharomyces cerevisiae mutants depleted of the voltage dependent anion selective channel (YVDAC1) are still able to grow on a non-fermentable carbon source, a functional transport system in the outer mitochondrial membrane must exist to support the access of metabolites into mitochondria. It was assumed that the properties of the system could be inferred from the differences in the results observed between wild type and mutant mitochondria since no crucial differences in this respect between the two types of mitoplasts were observed. YVDAC1-depleted mitochondria displayed a highly reduced permeability of the outer membrane, which was reflected in increased values of K0.5(NADH) for respiration and K0.5(ADP) for triggering phosphorylating state as well as in delayed action of carboxyatractylate (CATR) in inhibition of phosphorylating state. The parameters were chosen to express the accessibility of the applied species to the intermembrane space. The passage of the molecules through the outer membrane depleted of YVDAC1 could be partially improved in the presence of bivalent cations (Mg2+, Ca2+), as in their presence lower values of the calculated parameters were obtained. The restrictions imposed on the transport of molecules through the YVDAC1-depleted outer membrane resulted in a competition between them for the access to the intermembrane space as measured by changes in parameters observed for a given species in the presence of another one. The competition was stronger in the absence of Mg2+ and depended on charge and size of transported molecules, as the strongest competitor was CATR and the weakest one--NADH. Thus, it can be concluded that the transport system functioning in the absence of YVDAC1 is modulated by bivalent cations and charge as well as size of transported molecules. Since an increased level of respiration due to the dissipation of delta psi causes an increase of K0.5(NADH) in both wild type and YVDAC1-depleted mitochondria it is concluded that a common property of YVDAC1 and the system functioning in YVDAC1-depleted mitochondria seems to be the dependence of the capacity on the level of mitochondrial respiration.