RESUMO
The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction. Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages. Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography. The expressed protein was identified by enzyme assay and mass spectrometry. The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer.
Assuntos
Genes de Protozoários , Giardia lamblia/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Metabolismo Energético , Giardia lamblia/enzimologia , Hidrolases , Dados de Sequência Molecular , Peso Molecular , Fosfotransferases (Aceptor do Grupo Carboxila)/biossíntese , Fosfotransferases (Aceptor do Grupo Carboxila)/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The influx and efflux of alanine and other amino acids was studied in trophozoites of Giardia intestinalis. Transport of L-[2,3-3H]alanine was used as the index of influx. On the basis of the competition of L-[2,3-3H]alanine uptake by analogues of alanine, the substrate specificity of the alanine transporter was determined. The transporter is an antiport. Influx of alanine or those analogues which inhibited alanine influx caused the efflux of intracellular alanine and a number of amino acids structurally related to alanine. Amino acids unrelated to alanine, such as glutamate, effluxed at a slow rate, and the efflux was not stimulated by extracellular alanine or alanine analogues. However, there was a subset of intracellular amino acids, the alanine subset comprising alanine, serine, glycine, and threonine, the efflux of which was stimulated by external alanine or alanine analogues. Direct measurement by amino acid analysis demonstrated intracellular accumulation of alanine analogues concomitant with the efflux of the alanine subset. These data indicate unequivocal evidence of exchange of intracellular alanine with extracellular alanine analogues, with a 1:1 molar stoichiometry. This is the first demonstration in G. intestinalis of the antiport function of an amino acid transporter.
