Synthesis of pyruvate: ferredoxin oxidoreductase and alcohol dehydrogenase E enzymes during Giardia intestinalis excystation
Keywords:
Giardia lamblia, cell differentiation, alcohol dehydrogenase
Abstract
Introduction. Giardia intestinalis is a unicellular parasite of worldwide distribution. It causes an intestinal illness known as giardiasis, and it is probably the earliest diverging eukaryotic microorganism. Previously, changes have been reported in the expression of mRNAs at several stages of the life cycle; however specific enzymatic activity changes have not been explored.Objective. The expression of pyruvate ferredoxin oxidoreductase (PFOR) and alcohol dehydrogenase E (ADHE) enzymes was measured in cyst and trophozoite stages, and during the excystation process.
Materials and methods. Recombinant proteins were generated for PFOR and ADHE to be used as antigens in the production of polyclonal antibodies for the detection of native proteins by Western Blot. The enzymatic activity of ADHE and glutamate dehydrogenase (GDH) was evaluated by spectrophotometric assays.
Results. PFOR (139 kDa) and ADHE (97 kDa) proteins were detected in trophozoites, but not in cysts. During excystation, ADHE protein was detected after the first phase of induction, but the PFOR protein appeared only after the second phase. This indicated that both proteins were synthesized during excystation, although at different times. ADHE enzymatic activity was present only in trophozoites and not in cysts whereas GDH activity was detected in both stages.
Conclusion. These results conclusively showed that PFOR and ADHE enzymes were translated during the excystation process and is strong evidence that active protein synthesis was occurring during excystation.
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References
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2. Hernandez P, Wasserman M. Do genes from the cholesterol synthesis pathway exist and express in Giardia intestinalis? Parasitol Res. 2006;98:194-9.
3. Hetsko L, McCaffery J, Svärd S, Meng T, Que X, Gillin F. Cellular and transcriptional changes during excystation of Giardia lamblia in vitro. Exp Parasitol. 1998;88:172-83.
4. Niño C, Wasserman M. Transcription of metabolic enzyme genes during the excystation of Giardia lamblia. Parasitol Int. 2003;52:291-8.
5. Luján H, Mowatt M, Byrd L, Nash T. Cholesterol starvation induces differentation of the intestinal parasite Giardia lamblia. Proc Natl Acad Sci USA. 1996;93:7628-33.
6. Gillin F, Reiner D, McCaferry J. Cell Biology of the primitive eukaryote Giardia lamblia. Annu Rev Microbiol. 1996; 50:679-705.
7. Thompson R, Reynoldson J, Mendis A. Giardia and giardiasis. Adv Parasitol. 1993;32:71-160.
8. Paget T, Macechko P, Jarroll E. Metabolic changes in Giardia intestinalis during differentiation. J Parasitol. 1998;84:222-6.
9. Lindmark D. Energy metabolism of the anaerobic protozoon Giardia lamblia. Mol Biochem Parasitol. 1980;1:1-12.
10. Müller M. Energy metabolism of protozoa without mitochodria. Annu Rev Microbiol. 1998;42:465-88.
11. Townson S, Upcroft J, Upcroft P. Characterisation and purification of pyruvate: ferredoxin oxidoreductase from Giardia lamblia. Mol Biochem Parasitol. 1996;79:183-93.
12. Sanchez L. Aldehyde dehydrogenase (CoA-acetylating) and the mechanism of ethanol formation in the amitochondriate protist Giardia lamblia. Arch Biochem Biophys. 1998; 354:57-64.
13. Keister D. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg. 1983;77:487-8.
14. Kane A, Waed H, Keusch G, Pereira M. In-vitro encystations of Giardia lamblia: large-scale production of in-vitro encystations efficiency. J Parasitol. 1991;177:974-81.
15. Bingham A, Meyer E. Giardia excystation can be induced in vitro in acidic solutions. Nature. 1979;227:301-2.
16. Coligan J, Dunn B, Ploegh H, Speicher D, Wingfield P. Current protocols in protein science. New York: John Wiley and Sons, Inc.; 1997.
17. Harlow E, Lane D. Antibodies a laboratory manual. New York: Cold Spring Harbour Laboratory; 1998.
18. Park J, Schofield P, Edwards M. Giardia intestinalis: Characterization of a NADP-dependent glutamate dehydrogenase. Exp Parasitol. 1998;88:131-8.
How to Cite
1.
Niño CA, Wasserman M. Synthesis of pyruvate: ferredoxin oxidoreductase and alcohol dehydrogenase E enzymes during Giardia intestinalis excystation. biomedica [Internet]. 2010 Mar. 1 [cited 2024 May 18];30(1):32-8. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/150
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