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Related Experiment Videos

Nitric oxide synthase. Structural studies using anti-peptide antibodies

V Riveros-Moreno1, C Beddell, S Moncada

  • 1Wellcome Research Laboratories, Beckenham, England.

European Journal of Biochemistry
|August 1, 1993
PubMed
Summary

Computer analysis predicted immunogenic regions of rat brain nitric oxide (NO) synthase. Antibodies raised against these peptides recognized the native enzyme, with varying success based on peptide characteristics, and showed limited cross-reactivity with inducible NO synthase.

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Area of Science:

  • Biochemistry
  • Immunology
  • Molecular Biology

Background:

  • Nitric oxide (NO) synthase is crucial in various physiological processes.
  • Understanding the structure and function of NO synthase isoforms is essential for therapeutic development.
  • Antibodies are valuable tools for protein localization and functional studies.

Purpose of the Study:

  • To characterize antibodies raised against synthetic peptides of constitutive rat brain NO synthase.
  • To compare the immunogenicity and antibody recognition of constitutive and inducible NO synthase isoforms.
  • To investigate the relationship between peptide physicochemical properties and antibody recognition of native NO synthase.

Main Methods:

  • Bioinformatic analysis of amino acid sequences to predict peptide properties (hydrophilicity, accessibility, etc.).

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  • Chemical synthesis of selected peptides and coupling to carrier proteins.
  • Antibody production in rabbits and characterization using Western blot and enzyme activity assays.
  • Immunological comparison between constitutive (rat brain) and inducible (macrophage) NO synthase.

    • Main Results:

    • Peptides predicted to be exposed and flexible generated antibodies that recognized native NO synthase.
    • Antibodies against peptides from potential binding sites showed low recognition of tertiary structure but were immunogenic.
    • Anti-peptide antibodies demonstrated limited cross-reactivity with inducible NO synthase, unlike antibodies against the whole brain enzyme.
    • Enzyme activity neutralization by anti-peptide antibodies suggested steric hindrance affecting antibody accessibility to binding sites.

    Conclusions:

    • Physicochemical properties of peptides influence antibody recognition of native NO synthase.
    • Steric hindrance may limit antibody access to substrate binding sites in NO synthase.
    • Constitutive and inducible NO synthase isoforms exhibit distinct antigenic properties.
    • Developed antibodies are specific tools for studying NO synthase localization and function.