digitonin 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

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

1. The production of pyruvate, glycerol and glycerol-3-phosphate by intact and digitonin-permeabilized Trypanosoma brucei brucei has been studied with glucose or the glycolytic intermediates as substrates. 2. Under aerobic conditions hexosephosphates gave maximal glycolysis in the presence of 40-60 micrograms digitonin/10(8) trypanosomes while the triosephosphates gave it at 20-30 micrograms digitonin/10(8) trypanosomes. 3. In the presence of salicylhydroxamic acid, and the glycolytic intermediates, permeabilized trypanosomes produced equimolar amounts of pyruvate and glycerol-3-phosphate and no glycerol. Under the same conditions, glucose catabolism produced glycerol in addition to pyruvated and glycerol-3-phosphate. 4. In the presence of salicylhydroxamic acid and ATP or ADP intact trypanosomes produced equimolar amounts of pyruvate and (glycerol plus glycerol-3-phosphate) with glucose as substrate. 5. A carrier for ATP and ADP at the glycosomal membrane is implicated. 6. It is apparent that glycerol formation is regulated by the ATP/ADP ratio and that it needs intact glycosomal membrane and the presence of glucose.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anaerobiosis; Animals; Cell Membrane Permeability; Digitonin; Glucose; Glycerol; Glycerophosphates; Glycolysis; Pyruvates; Rats; Salicylamides; Trypanosoma brucei brucei; Trypanosomiasis, African