digitonin and fructose-6-phosphate

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

After protein cross-linking by dimethyl suberimidate, tumoral insulin-producing cells of the RINm5F line were either exposed to digitonin for measurement of hexokinase activity in the resulting cell pellet and supernatant, or incubated in the presence of D-[5-3H]glucose, D-[U-14C]glucose or L-[U-14C]glutamine to assess the metabolism of these nutrients. After digitonin treatment, the activity of hexokinase recovered in the cell pellet was about 40% higher in cross-linked than control RINm5F cells. Although failing to affect the metabolism of L-[U-14C]glutamine, and severely decreasing the oxidation of D-[U-14C]glucose, the cross-linking of proteins accentuated the increase in D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to acidic metabolites resulting from a rise in hexose concentration from 2.8 to 16.7 mM. The latter change represents a mirror image of that previously found in cross-linked pancreatic islets. Taking into account the vastly different participation of glucokinase to hexose phosphorylation in RINm5F cells and normal islet cells, the present findings further support, therefore, the regulatory role of protein-to-protein interaction in the control of glucokinase catalytic activity in these fuel-sensing cells.

Topics: Animals; Cross-Linking Reagents; Digitonin; Dimethyl Suberimidate; Fructosephosphates; Glucose; Glucose-6-Phosphate; Glutamine; Hexokinase; Insulinoma; Rats; Tumor Cells, Cultured