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.
Department of Genetics, Biology and Biochemistry, University of Torino, Italy. dario.ghigo@unito.it
Dario
D
Ghigo
Chiara
C
Riganti
Elena
E
Gazzano
Costanzo
C
Costamagna
Amalia
A
Bosia
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