oligomycins and salicylhydroxamic-acid

Glycolysis in bloodstream T. brucei is the sole source of energy and remains a favourable chemotherapeutic target. In furtherance of this, an attempt has been made to understand better the contribution of glucose, fructose, mannose and glycerol to the energy charge of these parasites incubated in the presence of oligomycin, salicyhydroxamic acid (SHAM) and digitonin. Their cellular energy charge, when catabolizing glucose was 0.860, and under inhibition by oligomycin (10 microg), SHAM (2 mM) or oligomycin plus SHAM, 0.800, 0.444 and 0.405, respectively. Oligomycin inhibited the rate of catabolism of glucose, mannose and fructose up to 80%. The inhibition could not be alleviated by uncouplers, such as 2,4-dinitrophenol or permeabilization of the membranes by digitonin. Glucose-6-phosphate and other phosphorylated glycolytic intermediates, such as fructose-6-phosphate were catabolized by the permeabilized parasites in the presence of oligomycin, implying that except hexokinase, all the other glycolytic enzymes were active. Glucose oxidation was stimulated by low concentrations of digitonin (up to 4 microg), but at higher concentrations, it was significantly inhibited (up to 90% inhibition at 10 microg). Apparently, the inhibitory effects of oligomycin and digitonin were confined to glucose uptake and hexokinase catalysis. The above observations suggest that the hexose transporter and the enzyme hexokinase might be functionally-linked in the glycosomal membrane and oligomycin inhibits the linkage, by using a mechanism not linked to the energy charge of the cell. Digitonin at concentrations higher than 4 microg disrupted the membrane, rendering the complex in-operative. A hexokinase/hexose transporter complex in the glycosomal membrane is envisaged.

Topics: Adenosine Triphosphate; Animals; Ca(2+) Mg(2+)-ATPase; Digitonin; Dose-Response Relationship, Drug; Fructosephosphates; Glucose-6-Phosphate; Hexokinase; Hexoses; Microbodies; Monosaccharide Transport Proteins; Oligomycins; Salicylamides; Temperature; Time Factors; Trypanosoma brucei brucei

This article describes the first detailed analysis of mitochondrial electron transfer and oxidative phosphorylation in the pathogenic filamentous fungus, Gaeumannomyces graminis var. tritici. While oxygen consumption was cyanide insensitive, inhibition occurred following treatment with complex III inhibitors and the alternative oxidase inhibitor, salicylhydroxamic acid (SHAM). Similarly, maintenance of a Deltapsi across the mitochondrial inner membrane was unaffected by cyanide but sensitive to antimycin A and SHAM when succinate was added as the respiratory substrate. As a result, ATP synthesis through complex V was demonstrated to be sensitive to these two inhibitors but not to cyanide. Analysis of the cytochrome content of mitochondria indicated the presence of those cytochromes normally associated with electron transport in eukaryotic mitochondria together with a third, b-type heme, exhibiting a dithionite-reduced absorbance maxima at 560 nm and not associated with complex III. Antibodies raised to plant alternative oxidase detected the presence of both the monomeric and dimeric forms of this oxidase. Overall this study demonstrates that a novel respiratory chain utilizing the terminal oxidases, cytochrome c oxidase and alternative oxidase, are present and constitutively active in electron transfer in G. graminis tritici. These results are discussed in relation to current understanding of fungal electron transfer and to the possible contribution of alternative redox centers in ATP synthesis.

Topics: Antifungal Agents; Antimycin A; Ascomycota; Carboxin; Cytochromes; Electron Transport; Microbial Sensitivity Tests; Oligomycins; Oxygen Consumption; Potassium Cyanide; Salicylamides; Triticum

The catabolism of hexose sugars and glycerol by the bloodstream form of Trypanosoma brucei brucei incubated with oligomycin was investigated. Oligomycin at a concentration of 10 micrograms/10(8) trypanosomes inhibited the catabolism of fructose, glucose and mannose by 70-80%, but not that of glycerol. Permeabilization of the trypanosome membranes by digitonin did not reverse the inhibition by oligomycin. Oligomycin did not inhibit pyruvate production in digitonin-permeabilized trypanosomes which were catabolizing exogenous glycolytic intermediates. It is concluded that the oligomycin-sensitive glycolysis is dependent on trypanosome membrane integrity. Oligomycin caused a rapid increase in the levels of hexose phosphates and some triose phosphates, but a decrease in the levels of glycerate 2-phosphate and phosphoenolpyruvate. There was a crossover point in the sequence of reactions between the formation of glycerol 3-phosphate and glycerate 2-phosphate during catabolism of the hexoses. Addition of the same concentration of oligomycin caused no change in the levels of glycolytic intermediates during the catabolism of glycerol. It is proposed that the catabolism of hexose sugars requires the transport of glycerol 3-phosphate from the glycosome via a glycerol 3-phosphate carrier which is probably inhibited by a hexose-sugar derivative formed on inhibition of the mitochondrial Mg(2+)-ATPase by oligomycin.

Topics: Adenine Nucleotides; Animals; Cell Membrane Permeability; Digitonin; Glucose; Glycerol; Glycolysis; Hexoses; Oligomycins; Pyruvates; Pyruvic Acid; Rats; Rats, Wistar; Salicylamides; Trypanocidal Agents; Trypanosoma brucei brucei

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.

Topics: Animals; Energy Metabolism; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Oligomycins; Onium Compounds; Proton Pumps; Proton-Translocating ATPases; Protons; Rubidium; Salicylamides; Trityl Compounds; Trypanocidal Agents; Trypanosoma brucei brucei

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.

Topics: Adenosine Triphosphate; Blastomyces; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cysteine; Electron Transport; Mitochondria; Mitosporic Fungi; Oligomycins; Oxidative Phosphorylation; Oxygen Consumption; Paracoccidioides; Potassium Cyanide; Salicylamides; Temperature