Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli
| Autor: | Paulus Michels, L. Kohl, T Drmota, J. Van Beeumen, Mia Callens, Frederik Opperdoes, C D Thi |
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| Rok vydání: | 1996 |
| Předmět: |
Leishmania mexicana
Molecular Sequence Data Trypanosoma brucei brucei Gene Expression Sequence Homology Dehydrogenase Glycerolphosphate Dehydrogenase Biology Trypanosoma brucei Glycosome Complementary DNA parasitic diseases Animals Genomic library Amino Acid Sequence Cloning Molecular Molecular Biology Gene Peroxisomal targeting signal Phylogeny Glycerol-3-Phosphate Dehydrogenase (NAD+) Base Sequence Chromosome Mapping biology.organism_classification NAD Molecular biology Biochemistry Parasitology |
| Zdroj: | Molecular and biochemical parasitology. 76(1-2) |
| ISSN: | 0166-6851 |
| Popis: | A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed. |
| Databáze: | OpenAIRE |
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