RESUMO
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.
Assuntos
Glicerolfosfato Desidrogenase/química , Leishmania mexicana/enzimologia , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Expressão Gênica , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/genética , Leishmania mexicana/genética , Dados de Sequência Molecular , NAD/metabolismo , Filogenia , Homologia de Sequência , Trypanosoma brucei brucei/genéticaRESUMO
The gene coding for the glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana has been cloned into vector pET3A and expressed as a soluble and active protein in Escherichia coli BL21(DE3) in which the endogenous gene has been inactivated by mutation. The recombinant enzyme was purified to near homogeneity by ammonium sulphate precipitation, followed by hydrophobic and cation-exchange chromatography. From a 1-L culture of E. coli cells, 25 mg purified protein was obtained with a specific activity of 125 units/mg. The recombinant protein restores the natural E. coli phenotype when expressed at low level. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. The recombinant and the native proteins show identical mobilities on SDS/PAGE, and have the same isoelectric point and similar pH-activity profiles. The kinetics of both enzymes are very similar, the most important aspect being their lower apparent affinity for the cofactor NAD when compared to all other homologous enzymes studied, with the exception of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Leishmania mexicana/enzimologia , Organelas/enzimologia , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Indução Enzimática , Eritrócitos/enzimologia , Escherichia coli , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Humanos , Concentração de Íons de Hidrogênio , Cinética , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/enzimologiaRESUMO
The gene of triose-phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27,561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody-like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triose-phosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues constituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme. L. mexicana triose-phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3-phosphate as substrate Km = 0.30 +/- 0.05 mM and kcat = 2.5 x 10(5) min-1; with dihydroxyacetone phosphate as substrate Km = 1.3 +/- 0.3 mM and kcat = 2.8 x 10(4) min-1.
Assuntos
Leishmania mexicana/enzimologia , Leishmania mexicana/genética , Triose-Fosfato Isomerase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Cromatografia por Troca Iônica , Clonagem Molecular/métodos , Primers do DNA , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Ligação de Hidrogênio , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Triose-Fosfato Isomerase/isolamento & purificação , Triose-Fosfato Isomerase/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genéticaRESUMO
Leishmania mexicana mexicana contains two tandemly arranged genes for pyruvate kinase (PYK). The 5' located gene codes for a polypeptide with a molecular mass of 54,370. The calculated net charge and isoelectric point of the polypeptide are -6 and 6.5, respectively. Its amino-acid sequence is 73.7% identical to that of the Trypanosoma brucei PYK and 46.4-49.8% to the enzyme of mammalian cells. The second gene appears not to be functional, because its 5' and 3' extremities have undergone recombinations. L. m. mexicana PYK has been overexpressed in Escherichia coli, using a T7 expression system. Approximately 30% of the protein was detected in the soluble cell fraction. It has been highly purified by chromatography over DEAE-Sephacel and Affigel Blue. From a 1-1 culture 6 mg enzyme was obtained with a specific activity of 224 units mg-1. The protein has a subunit molecular mass of 59,000, as determined by SDS/PAGE, and an isoelectric point of 5.9. Some kinetic properties of the enzyme have been measured and compared with those reported for the T. brucei enzyme. The kinetics of both enzymes are very similar, the most important aspect being their activation by fructose 2,6-bisphosphate. Nevertheless, some differences were observed; the T. brucei enzyme is activated by the effector in a cooperative manner, whereas the activation of the L. m. mexicana enzyme is not cooperative.