Toxoplasma gondii: molecular cloning and characterization of a nitric oxide synthase-like protein.
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.
Gutierrez-Escobar
Andrés J
AJ
Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia.
Arenas
Aylan Farid
AF
Villoria-Guerrero
Yanet
Y
Padilla-Londoño
Jonathan M
JM
Gómez-Marin
Jorge Enrique
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003124:Colorimetry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics
D019001:Nitric Oxide Synthase