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

Using an oligonucleotide primer based on a partial goldfish inducible nitric oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated from an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1,127-amino acid protein 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 NADPH. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Inducibility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or the protozoan blood flagellate Trypanoplasma borreli. These stimulators produced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transcription factor NF-kappaB was shown to play a role in the induction of iNOS transcription.