Molecular and functional characterization of a fish inducible-type nitric oxide synthase

Using an oligonucleotide primer based on a partial goldfish inducible nitri c oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated fr om an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1127-amino acid p rotein with 57% sequence identity to human iNOS. Like mammalian NOSs, carp iNOS protein contains putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADP H. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Induci bility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or t he protozoan blood flagellate Trypanoplasma borreli. These stimulators prod uced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transciption factor NF-kappa B was shown to play a role in the induction of iNOS transcription.