Njogu, MR;, Kiaira JK.  2004.  Inhibition of the hexokinase/hexose transporter region in the glycosomal membrane of bloodstream Trypanosoma brucei by oligomycin and digitonin. Abstract

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.


Olembo, NK, Obungu VH, Kiaira JK, Njogu RM.  1999.  Catabolism of proline by procyclic culture forms of Trypanosoma congolense. AbstractWebsite

The effect of various metabolic inhibitors on the rate of oxygen consumption by procyclic culture forms of Trypanosoma congolense utilizing proline as substrate was investigated. Cyanide inhibited the rate of oxygen consumption by 81.0 +/- 6.7%, malonate inhibited the rate by 51.6 +/- 1.6% and Antimycin A by 73.1 +/- 5.9%. A combination of cyanide and malonate inhibited the rate of oxygen consumption by 84.9 +/- 6.7% while a combination of antimycin A and malonate inhibited the rate by 81.6 +/- 7.6%. Rotenone had no effect on the rate of respiration except when the intact cells were first permeabilized by digitonin after which rotenone decreased the rate of respiration by 20-30%. Salicylhydroxamate (SHAM) did not have any effect on the rate of oxygen consumption. Enzymes involved in the catabolism of proline with high activities were: proline dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, NADP-linked malic enzyme, alanine aminotransferase and malate dehydrogenase. Activities of 1-pyrroline-5 carboxylate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and NAD-linked malic enzyme were detectable but lower. The end products of proline catabolism were alanine and glutamate. Unlike the case in Trypanosoma brucei brucei aspartate was not detected. Possible pathways of proline catabolism in procyclic culture forms of T. congolense and of electron transfer are proposed



Njogu, RM, Kiaira JK.  1988.  Trypanosoma brucei brucei: the catabolism of glycolytic intermediates by digitonin-permeabilized bloodstream trypomastigotes and some aspects of regulation of anaerobic glycolysis. Abstract

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.


Kiaira, JK, Njogu RM.  1983.  Comparison of glycolysis in intact and digitonin-permeabilized bloodstream trypomastigotes of Trypanosoma brucei. AbstractWebsite

Digitonin has been used to permeabilize bloodstream trypomastigotes of Trypanosoma brucei. Such permeabilized parasites revealed a fully-functional glycolytic pathway which catabolized glucose and some phosphorylated glycolytic intermediates. Glucose-starved bloodstream trypomastigotes revealed saturation kinetics with a glucose Km = 0.6 mM and Vmax = 150 natom O/min per 10(8) for intact parasites; Km = 4 mM and Vmax = 100 natom O2/min per 10(8) for permeabilized parasites. Glucose oxidation in intact parasites was stimulated 40% by addition of 3 micrograms digitonin/10(8) parasites. Higher concentrations of digitonin than this inhibited the glucose oxidation. Ten millimolar phosphoenolpyruvate (PEP) inhibited the rate of O2 consumption by permeabilized trypanosomes respiring on glucose under aerobic conditions by 50%. It is proposed that glucose oxidation is apparently limited by transport across trypanosomal plasma membrane, and phosphofructokinase is regulated by PEP levels. It is concluded that permeabilization of trypanosomes with digitonin might offer a closer physiological condition for the study of the regulation of glycolysis by using glycolytic intermediates and other chemical compounds which would otherwise not be transported across the membrane(s).


  1982.  Trypanosoma brucei: a quick method for separating blood-stream trypomastigotes from infected blood by differential osmotic lysis. Abstract

The degree of rat erythrocyte lysis and immobilization of Trypanosoma brucei in infected blood by buffered hypotonic solutions of sodium chloride and sources was studied. 2. At 0.3% sodium chloride solution 98% hemolysis of erythrocytes was achieved while 95% of the original bloodstream trypomastigotes survived and were found to be motile and viable for biochemical study. 3. Further increase in the concentration of sodium chloride above 0.3% revealed an increase in the immobilization of trypanosomes and a decrease in the erythrocyte hemolysis. 4. Bloodstream trypomastigotes have been prepared by differential osmotic lysis of infected blood in 0.3% sodium chloride solution and used for studying their metabolism.


Njogu, RM, Nyindo M.  1981.  Presence of a peculiar pathway of glucose metabolism in infective forms of Trypanosoma brucei cultured from salivary glands of tsetse flies. Abstract

We have studied metabolism of glucose by infective forms of Trypanosoma brucei brucei isolated from tsetse fly salivary glands and grown in continuous culture for more than 700 days. The end products of glucose metabolism under aerobic conditions were found to be pyruvate and glycerol in the ratio 3:1. this changed to equimolar formation of pyruvate and glycerol when glucose was metabolized under aerobic conditions in the presence of 1.5 mM salicylhydroxamic acid (SHAM), a situation analogous to anaerobiosis. The respiration of the parasites was insensitive to cyanide inhibition but SHAM inhibited it completely. In the presence of SHAM, formation of pyruvate and glycerol from glucose metabolism was inhibited by 60% and 45% respectively. These findings indicate that the infective trypanosomes grown in vitro for about 2 yr have a peculiar glycolytic pathway which can be exploited to elucidate the mechanism of energy production of bloodstream form trypanosomes during anaerobic glycolysis


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