Cycling of NADPH by glucose 6-phosphate dehydrogenase optimizes the spectrophotometric assay of nitric oxide synthase activity in cell lysates

Dario Ghigo; Chiara Riganti; Elena Gazzano; Costanzo Costamagna; Amalia Bosia

doi:10.1016/j.niox.2006.01.002

Cycling of NADPH by glucose 6-phosphate dehydrogenase optimizes the spectrophotometric assay of nitric oxide synthase activity in cell lysates

Nitric Oxide. 2006 Sep;15(2):148-53. doi: 10.1016/j.niox.2006.01.002. Epub 2006 Feb 17.

Authors

Dario Ghigo¹, Chiara Riganti, Elena Gazzano, Costanzo Costamagna, Amalia Bosia

Affiliation

¹ Department of Genetics, Biology and Biochemistry, University of Torino, Italy. dario.ghigo@unito.it

PMID: 16483808
DOI: 10.1016/j.niox.2006.01.002

Abstract

The measurement of nitric oxide synthase activity in cell lysates is often performed by radiochemical assay that quantifies 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 sufficient to be detected with the Griess assay. The incorporation of cycling of NADPH also improves the radiochemical assay for nitric oxide synthase activity.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Biological Assay / methods*
Cell Extracts / chemistry
Cells, Cultured
Glucosephosphate Dehydrogenase / chemistry*
Mice
NADP / chemistry*
Nitric Oxide Synthase / analysis*
Spectrophotometry / methods*

Substances

Cell Extracts
NADP
Glucosephosphate Dehydrogenase
Nitric Oxide Synthase