Cycling of NADPH by glucose 6-phosphate dehydrogenase optimizes the spectrophotometric assay of nitric oxide synthase activity in cell lysates.
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.
Ghigo
Dario
D
Department of Genetics, Biology and Biochemistry, University of Torino, Italy. dario.ghigo@unito.it
Riganti
Chiara
C
Gazzano
Elena
E
Costamagna
Costanzo
C
Bosia
Amalia
A
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry*; D051379:Mice; D009249:NADP / Q000737:chemistry*; D019001:Nitric Oxide Synthase / Q000032:analysis*; D013053:Spectrophotometry / Q000379:methods*
D002457:Cell Extracts; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D019001:Nitric Oxide Synthase