RESUMO

Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of l-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis-Menten kinetics, with a K(m) for L-arginine of 2.3mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from L-arginine as substrate.

Assuntos

Óxido Nítrico Sintase/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Colorimetria , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/isolamento & purificação , Nucleopoliedrovírus/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spodoptera , Toxoplasma/genética