The measurement of nitric oxide synthase activity in cell lysates is often performed by radiochemical assay that quantiWes the conversion of L-[3H]arginine to L-[3H]citrulline. We have developed a spectrophotometric procedure which continuously recycles NADPH through the addition of glucose 6-phosphate dehydrogenase to the cell lysate. This allows nitric oxide synthase to operate linearly for hours, so that nitric oxide-derived nitrite accumulates at amounts suYcient to be detected with the Griess assay. The incorporation of cycling of NADPH also improves the radiochemical assay for nitric oxide synthase activity.
Metodo di misura dell'attività dell'enzima ossido nitrico sintasi (NOS)
GHIGO, Dario Antonio;RIGANTI, Chiara
2004-01-01
Abstract
The measurement of nitric oxide synthase activity in cell lysates is often performed by radiochemical assay that quantiWes the conversion of L-[3H]arginine to L-[3H]citrulline. We have developed a spectrophotometric procedure which continuously recycles NADPH through the addition of glucose 6-phosphate dehydrogenase to the cell lysate. This allows nitric oxide synthase to operate linearly for hours, so that nitric oxide-derived nitrite accumulates at amounts suYcient to be detected with the Griess assay. The incorporation of cycling of NADPH also improves the radiochemical assay for nitric oxide synthase activity.
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