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Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)
Published on: April 6, 2019
Transketolase in Trypanosoma brucei.
Sabine A Stoffel1, Vincent P Alibu, Jane Hubert
1Pevion Biotech AG, Worblentalstrasse 32, CH-3063 Ittigen/BE, Switzerland.
Molecular and Biochemical Parasitology
|May 17, 2011
Summary
A Trypanosoma brucei transketolase gene was identified and expressed, showing dual cytosolic and glycosomal localization. While essential for procyclic forms, its absence in bloodstream forms suggests differential regulation, with no observed growth phenotype in null mutants.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Transketolase (TKT) is a key enzyme in the pentose phosphate pathway.
- Trypanosoma brucei exhibits distinct life cycle stages with unique metabolic requirements.
Purpose of the Study:
- To characterize the TKT gene and protein in Trypanosoma brucei.
- To investigate the enzyme's subcellular localization and expression patterns.
- To determine the physiological role of TKT in T. brucei.
Main Methods:
- Gene identification and expression in E. coli.
- Enzyme activity assays and kinetic analysis (K(m) determination).
- Subcellular localization studies using T. brucei.
- Gene knockout and metabolite profiling.
Main Results:
- A TKT gene was identified in T. brucei, with the purified protein exhibiting enzymatic activity.
- The TKT protein displayed dual localization in both glycosomes and the cytosol.
- TKT expression was predominantly in procyclic forms, absent in bloodstream forms, indicating differential regulation.
- TKT knockout mutants showed no growth defect but altered metabolite profiles, with increased substrates and decreased product (sedoheptulose 7-phosphate).
Conclusions:
- TKT plays a significant role in the procyclic stage of T. brucei, likely in carbohydrate metabolism.
- The dual localization and differential expression suggest complex regulatory mechanisms.
- Despite metabolic alterations, TKT is not essential for growth in vitro, highlighting metabolic flexibility or alternative pathways.
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