Exp Parasitol. 2008 Mar 23 [Epub ahead of print]
Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein
Gutierrez-Escobar AJ, Arenas AF, Villoria-Guerrero Y, Padilla-Londoño JM, Gómez-Marin JE.
Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia.
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 21nmol/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 11.6kDa 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.
PMID: 18439581 [PubMed - as supplied by publisher]