NITRIC-OXIDE SYNTHASE - STRUCTURAL STUDIES USING ANTIPEPTIDE ANTIBODIES

The amino acid sequence for the constitutive rat brain nitric oxide (NO) synthase was analysed by a set of computer programs that estimate and display physicochemical properties such as hydrophilicity, flexibility, accessibility, hydrophilic periodicity and conformation [Comerford, S. A., McCance, D. J., Dougan, G. & Tite, J. P. (1991) J. Virol. 65, 4681-4690]. Overall, they allow prediction of whether each peptide region will be an alpha-helix, a beta-strand or a less regular coil and also whether the region will be buried in the protein core or exposed to water at the surface of the protein molecule. Ten peptide regions were chosen; the majority were predicted to be exposed areas of the molecule and therefore likely to be immunogenic. The peptides were chemically synthesised, coupled to keyhole limpet haemocyanin carrier protein and injected into rabbits to raise antibodies. These antibodies have been used by us and others to locate the NO synthase in different tissues and species. Here we present the characterisation of the antibodies in relation to the possible conformation of the enzyme and an immunological comparison between two isoforms of NO synthase: constitutive (rat brain) and inducible (macrophage). Peptide regions predicted to be exposed, flexible or substantially in core, have produced antibodies that were able to recognise the native protein. Peptides of mixed characteristics possibly involved in the binding site tended to produce antibodies with low recognition for the tertiary structure of the native, purified NO synthase, although these peptides were all highly immunogenic. We postulate that either the peptides when conjugated to the carrier protein attain a different conformation to that in the native NO synthase, or alternatively the accessibility of the antibodies to substrate binding sites is highly restricted by steric hindrance. This latter seems to be more likely since a mixture of antibodies against this area of the protein molecule was able to achieve a similar neutralisation of the enzyme activity as the antibodies against the whole enzyme (almost-equal-to 50%). Most of the selected anti-peptide antibodies were not able to cross-react with the inducible macrophage enzyme; only two that have 60% sequence identity showed a weak reaction in Western blot. The polyclonal antibody against the complete brain enzyme showed cross-reaction in a Western blot with inducible enzyme. The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required. We propose that conformational epitopes are responsible for the cross-reaction, suggesting that the overall tertiary structures of the two enzymes share a degree of similarity.