Wilson, Natalie Jane; (2000) Molecular and biochemical characterisation of a Fugu nitric oxide synthase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A neuronal nitric oxide synthase (NOSl) gene was isolated from the the model vertebrate genome of the Japanese pufferfish Fugu rubripes. The Fugu NOSl gene was cloned by screening of a genomic cosmid library and PCR of genomic DNA. It was sequenced multiple times on both strands and conserved co-factor/substrate binding sites for haem, L-arginine, Ca2+/Calmodulin, FAD, FMN and NADPH were identifed. In addition, a CaM inhibitory sequence present only in constitutive isoforms was identified along with a PDZ domain specific to nNOS isoforms. At 22,203 bp the Fugu NOSl gene shows a reduction in size of -12 times when compared to the human NOSl gene. Full-length Fugu NOSl cDNA (fNOS) was isolated from the brain of another species of Japanese pufferfish, Takifugu poecilonotus, by 5' and 3' RACE and PCR. fNOS was cloned into the baculovirus-transfer vector pVL1393, sequenced and transfected into Spodoptera frugiperda clone 21 cells (Sf21) for expression studies. fNOS showed the highest amino acid identity to the mammalian and Xenopus laevis nNOS isoforms (74–75%). Amino acid identity dropped to 58% when fNOS was compared to eNOS isoforms, 54% for iNOS isoforms and 47–52% for the reported invertebrate NOS isoforms. Expression of the recombinant Fugu NOSl protein (FNOS) by pVLfNOS- infected Sf21 cells was detected by western blotting and immunocytochemical staining with a monoclonal anti-nNOS antibody. Expression of functional FNOS was greatest after 24 hours as measured by the spectrophometric conversion of oxyhaemoglobin to methaemoglobin by NO on a dual-wavelength spectrophotometer. In the presence of FAD, NADPH, and BH4, FNOS activity was dependent on the addition of L-arginine and was inhibitable by the NOS inhibitors L-NMMA, L-Thiocitrulline and 1400W. Partial purification of FNOS was achieved using a 2'5' ADP sepharose column and lOmM NADPH for elution.

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