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Neuronal NADPH diaphorase is a nitric oxide synthase.
B T Hope1, G J Michael, K M Knigge
1Kinsmen Laboratory of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, Canada.
Summary
NADPH diaphorase histochemistry identifies neurons producing nitric oxide. This study biochemically confirms neuronal NADPH diaphorase is a nitric oxide synthase, validating its use as a marker.
Area of Science:
- Neuroscience
- Biochemistry
- Enzymology
Background:
- NADPH diaphorase histochemistry is a widely used technique to label neuronal populations.
- The precise function of NADPH diaphorase in the nervous system has been unclear.
- Nitric oxide synthase (NOS) is a 150-kDa NADPH-dependent enzyme in the brain involved in nitric oxide synthesis.
Purpose of the Study:
- To biochemically characterize NADPH diaphorase.
- To determine if NADPH diaphorase is related to nitric oxide synthase.
Main Methods:
- Purification of NADPH diaphorase from rat brain using affinity chromatography and HPLC.
- Western immunoblotting and immunostaining to identify protein size.
- Biochemical assays to assess enzyme activity and inhibition.
- Immunoprecipitation studies.
Main Results:
- NADPH diaphorase was purified to homogeneity, yielding a single 150 kDa protein.
- NADPH diaphorase activity and nitric oxide synthase activity copurified.
- Both activities were immunoprecipitated by an antibody against neuronal NADPH diaphorase.
- Nitric oxide synthase activity was competitively inhibited by a NADPH diaphorase substrate.
Conclusions:
- Neuronal NADPH diaphorase is identical to nitric oxide synthase.
- NADPH diaphorase histochemistry serves as a specific marker for neurons that produce nitric oxide.