Determination of nitrite by high-performance liquid chromatography system with electrochemical detector: Measurement of nitric oxide synthase activity in rat cerebellum cytosol
✍ Scribed by Shinsuke Kaku; Makoto Tanaka; Makoto Muramatsu; Susumu Otomo
- Publisher
- John Wiley and Sons
- Year
- 1994
- Tongue
- English
- Weight
- 487 KB
- Volume
- 8
- Category
- Article
- ISSN
- 0269-3879
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✦ Synopsis
A simple and sensitive assay method for NO synthase activity is described. Using glassy carbon as electrode and 30% methanol solution with 10mM NH&I as mobile phase, NO, can be measured without disturbing ECD-detectable substance in NO synthase assay mixture. The NO; production in the assay mixture of rat cerebellum NO synthase increased with protein and in a time-dependent manner. The K , value for the substrate, barginbe, was 1.25 PM. The enzyme activity was inhibited in a concentration-dependent manner by a NO synthase inhibitor, NNA. The Ki value for NNA was 0.166t0.060 PM. This ECD-HPLC method for determining NO synthase activity has advantages compared with the diazo-coupling method of the Greiss reagent and the isotopic method in which the conversion of the substrate, ['4C]r,-arginine, to the product, ['~]~-citrulline is measured; it is simple, sensitive and is convenient for studying the NO synthase activity with various compounds as the substrate.