Complete coding sequence of equine inducible nitric oxide synthase.

Abstract: THE gene encoding equine inducible nitric oxide synthase (iNOS) was cloned and sequenced. The strategy consisted of the amplification by PCR of message isolated from primary cultures of equine articular chondrocytes (Frean et al 1997) treated with 100 mg mlÿ1 LPS (Escherichia coli serotype O127:B8). PCR primer pairs were generated by comparison of cross-species published iNOS nucleotide sequences. The sequences of the 30 and 50 termini were derived by the generation of PCR products spanning those regions. The assembly of sequenced PCR fragments revealed a 3930 complete nucleotide sequence with a single open reading frame beginning with an ATG methionine codon in position 1 and ending at position 3930 with a TAA stop codon. The 3930 base pair (bp) open reading frame encodes a predicted polypeptide of 1310 amino acids with a calculated molecular mass of 148 kDa and a predicted pI of 8.6. Presumptive FAD, FMN and NADPH binding regions were predicted on the basis of those shown to be characteristic for iNOS in previous reports (Forstermann et al 1994). Sequence data have been deposited in the GenBank database (Accession No. AY027883). Detection of iNOS mRNA by Northern blot was used to confirm the validity of the sequenced fragment as being part of the iNOS cDNA. The calculated molecular mass for equine iNOS is greater than that reported for iNOS in other species (Forstermann et al 1994). Comparison of published iNOS sequences demonstrates a 79 per cent identity between the equine and canine nucleotide sequence. At the protein level, identity between human, dog and equine sequences is 72 to 73 per cent. The cofactor binding regions are characteristic for nitric oxide synthases (Bredt et al 1991) and show greater conservation of sequence between species (Adams et al 1998). Our study shows a higher degree of cofactor site conservation for FAD (92 per cent), FMN (96 per cent) and calmodulin (75 per cent) between human and equine iNOS than for human and mouse iNOS (84, 93 and 66 per cent respectively). In contrast, the NADPH1 site in iNOS is better conserved between mouse and human (94 per cent) than between human and horse (83 per cent). The NADPH2 site is 87 per cent conserved between all three species. Such differences in the cofactor regions of the protein might alter the catalytic efficiency of the iNOS enzyme and may explain why the release of NO from equine, human and murine cells is so different.