Nitric oxide synthase

Abstract

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, 4681-46901. Overall, they allow prediction of whether each peptide region will be an a-helix, a P-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 (= 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.Nitric oxide (NO) synthase has been the subject of intensive study, focusing on the mode of action Stuehr, 1992;Bredt et al., 1992), distribution in different tissues, organs and species (Salter et al., 1991 ;Springall et al., 1992;Bredt et al., 1990), as well as comparisons between the enzymes at the molecular lev...

Localization of Nitric-oxide Synthase in Plant Peroxisomes

“…To rule out the possibility that the cross-reactivity observed is due to NADPH-cytochrome P450 reductases, a polyclonal antibody against 14 residues from the C terminus of iNOS which are absent in the NADPH-cytochrome P450 reductases, was used. This type of anti-peptide antibodies provide powerful tools to discriminate among proteins with a high degree of homology but with differences in other regions of the sequence (37). In this way, an immunoreactive band with a molecular mass of approximately 130 kDa was detected, which is in the same molecular mass range described for most iNOS (38,39) and eNOS (40,41) but different from the molecular mass reported for NADPH-cytochrome P450 reductases (70 kDa) (4,42).…”

Nitric oxide synthase in the Guinea pig preoptic area and hypothalamus: Distribution, effect of estrogen, and colocalization with progesterone receptor

“…Polak, London, U.K.) was used at a dilution of 1:1,000. NOS was purified from whole rat brain according to the method of Bredt and Snyder (1990) and injected into rabbits as described elsewhere (Springall et al, 1992;Riveros-Moreno et al, 1993). This antiserum has been shown to react immunocytochemically with neurons from different species (Springall et al, 1992;Terenghi et al, 1993;Martinez et al, 1994).…”

Detection of nitric oxide synthase (NOS) in somatostatin-producing cells of human and murine stomach and pancreas.

“…Kent, UK). The amino acid sequence of this peptide was LPLIQANGNDPELFQIPPELC, as described by Riveros-Moreno et al (15). The optimal dilution for the antiserum was 1:1000.…”