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[In vitro decrease of the cytolytic effect of E. histolytica by inhibition of its phosphofructokinase]
E Jiménez Cardoso1, E Cuevas Rosas, J M Jiménez Cardoso
1Laboratorio de Investigación en Parasitología, Hospital Infantil de México Federico Gómez, México D.F.
Abstract:
The C14 radioactive label of PPi analogues was incorporated to E. histolytica after 24 hours of incubation at 37 degrees C; more than 90% of trophozoites remained viable. The PPi dependent phosphofructokinase was isolated in order to determine its kinetic parameters. With PPi, the Km was 18.06 +/- 0.91 micromol/mL-1. Using three different PPi analogues (tetrasodium salts) of (I) 1,1 hydroxy-methyl diphosphonate; (II) 1,1 hydroxy ethylene diphosphonate; (III) 1,1 hydroxy-nonano diphosphonate, KiI was 35.19 +/- 1.74; KiII was 42.65 +/- 0.65, and KiIII 2as 62.81 +/- 0.27 micromol/mL-1. The graphic expression of these results shows that the enzyme was competitively inhibited by the three analogues. When trophozoites were incubated with each one of the three inhibitors, a correlation was observed between the concentration and the cytolytic inhibition with an r = 0.98. Nevertheless, the slope obtained was different for each one of them. The smallest concentration of inhibitor to achieve a 50% lysis inhibition of trophozoites was that of inhibitor III. In addition, it was demonstrated that the incubation of the trophozoites with this inhibitor increased the time needed to destroy CHO cells. We conclude that enzymatic inhibition of the PPi dependent phosphofructokinase caused by the PPi analogues was responsible for the modification of the lytic capacity of trophozoites, possibly by altering the metabolic pathway of carbohydrates.
Insights
Pyrophosphate (PPi) analogues competitively inhibit E. histolytica phosphofructokinase, reducing trophozoite cytolytic activity. Inhibitor III showed the greatest efficacy in reducing parasite lysis and protecting host cells.
Area of Science:
- Biochemistry
- Parasitology
- Enzymology
Context:
- Entamoeba histolytica (E. histolytica) is an anaerobic protozoan parasite responsible for amoebiasis.
- Phosphofructokinase (PFK) is a key enzyme in glycolysis, regulating carbohydrate metabolism.
- Understanding PFK inhibition in E. histolytica could reveal new therapeutic targets.
Purpose:
- To investigate the inhibitory effects of pyrophosphate (PPi) analogues on the PPi-dependent phosphofructokinase of E. histolytica.
- To determine the kinetic parameters of the enzyme and the inhibition constants (Ki) of the PPi analogues.
- To correlate enzyme inhibition with the parasite's cytolytic activity and its ability to damage host cells.
Summary:
- Radioactively labeled PPi analogues were incorporated into E. histolytica trophozoites, with over 90% viability maintained.
- The PPi-dependent phosphofructokinase was isolated, and kinetic parameters were determined. Three PPi analogues competitively inhibited the enzyme.
- Inhibitor III demonstrated the most potent inhibition, requiring the lowest concentration for 50% lysis inhibition and increasing the time for trophozoites to destroy host cells.
Impact:
- Enzymatic inhibition of PPi-dependent phosphofructokinase by PPi analogues modifies trophozoite lytic capacity.
- This inhibition likely alters carbohydrate metabolic pathways within the parasite.
- The findings suggest PPi analogues as potential agents to control E. histolytica infections by targeting its glycolysis.