The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression
NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr h...
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todo:paper_02646021_v330_n2_p951_Barderi2023-10-03T15:12:58Z The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression Barderi, P. Campetella, O. Frasch, A.C.C. Santomé, J.A. Hellman, U. Pettersson, U. Cazzulo, J.J. glutamate dehydrogenase nicotinamide adenine dinucleotide phosphate oligonucleotide proteinase trypsin amino acid sequence article carboxy terminal sequence epimastigote genome nucleotide sequence open reading frame priority journal protein expression trypanosoma cruzi Amino Acid Sequence Animals Base Sequence Cytosol DNA, Protozoan Escherichia coli Evolution, Molecular Glutamate Dehydrogenase (NADP+) Molecular Sequence Data Recombinant Proteins Trypanosoma cruzi Escherichia coli Eukaryota Protozoa Trypanosoma Trypanosoma cruzi NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme. Fil:Barderi, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Campetella, O. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_02646021_v330_n2_p951_Barderi |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
glutamate dehydrogenase nicotinamide adenine dinucleotide phosphate oligonucleotide proteinase trypsin amino acid sequence article carboxy terminal sequence epimastigote genome nucleotide sequence open reading frame priority journal protein expression trypanosoma cruzi Amino Acid Sequence Animals Base Sequence Cytosol DNA, Protozoan Escherichia coli Evolution, Molecular Glutamate Dehydrogenase (NADP+) Molecular Sequence Data Recombinant Proteins Trypanosoma cruzi Escherichia coli Eukaryota Protozoa Trypanosoma Trypanosoma cruzi |
| spellingShingle |
glutamate dehydrogenase nicotinamide adenine dinucleotide phosphate oligonucleotide proteinase trypsin amino acid sequence article carboxy terminal sequence epimastigote genome nucleotide sequence open reading frame priority journal protein expression trypanosoma cruzi Amino Acid Sequence Animals Base Sequence Cytosol DNA, Protozoan Escherichia coli Evolution, Molecular Glutamate Dehydrogenase (NADP+) Molecular Sequence Data Recombinant Proteins Trypanosoma cruzi Escherichia coli Eukaryota Protozoa Trypanosoma Trypanosoma cruzi Barderi, P. Campetella, O. Frasch, A.C.C. Santomé, J.A. Hellman, U. Pettersson, U. Cazzulo, J.J. The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression |
| topic_facet |
glutamate dehydrogenase nicotinamide adenine dinucleotide phosphate oligonucleotide proteinase trypsin amino acid sequence article carboxy terminal sequence epimastigote genome nucleotide sequence open reading frame priority journal protein expression trypanosoma cruzi Amino Acid Sequence Animals Base Sequence Cytosol DNA, Protozoan Escherichia coli Evolution, Molecular Glutamate Dehydrogenase (NADP+) Molecular Sequence Data Recombinant Proteins Trypanosoma cruzi Escherichia coli Eukaryota Protozoa Trypanosoma Trypanosoma cruzi |
| description |
NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme. |
| format |
JOUR |
| author |
Barderi, P. Campetella, O. Frasch, A.C.C. Santomé, J.A. Hellman, U. Pettersson, U. Cazzulo, J.J. |
| author_facet |
Barderi, P. Campetella, O. Frasch, A.C.C. Santomé, J.A. Hellman, U. Pettersson, U. Cazzulo, J.J. |
| author_sort |
Barderi, P. |
| title |
The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression |
| title_short |
The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression |
| title_full |
The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression |
| title_fullStr |
The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression |
| title_full_unstemmed |
The NADP+ -linked glutamate dehydrogenase from Trypanosoma cruzi: Sequence, genomic organization and expression |
| title_sort |
nadp+ -linked glutamate dehydrogenase from trypanosoma cruzi: sequence, genomic organization and expression |
| url |
http://hdl.handle.net/20.500.12110/paper_02646021_v330_n2_p951_Barderi |
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