Determination of nitrite by high-performance liquid chromatography system with electrochemical detector: measurement of nitric oxide synthase activity in rat cerebellum cytosol.
A simple and sensitive assay method for NO synthase activity is described. Using glassy carbon as electrode and 30% methanol solution with 10 mM NH4Cl as mobile phase, NO2- can be measured without disturbing ECD-detectable substance in NO synthase assay mixture. The NO2- production in the assay mixture of rat cerebellum: NO synthase increased with protein and in a time-dependent manner. The Km value for the substrate, L-arginine, was 1.25 microM. The enzyme activity was inhibited in a concentration-dependent manner by a NO synthase inhibitor, NNA. The Ki value for NNA was 0.166 +/- 0.060 microM. 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, [14C]L-arginine, to the product, [14C]L-citrulline is measured; it is simple, sensitive and is convenient for studying the NO synthase activity with various compounds as the substrate.
Kaku
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S
Department of Pharmacology, Taisho Pharmaceutical Co. Ltd., Saitama, Japan.
Tanaka
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M
Muramatsu
M
M
Otomo
S
S
D000594:Amino Acid Oxidoreductases / Q000032:analysis* / Q000037:antagonists & inhibitors; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002531:Cerebellum / Q000737:chemistry / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D008297:Male; D009566:Nitrates / Q000032:analysis*; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D009566:Nitrates; D019335:Nitroarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases