A Microtiter-Plate Assay of Human NOS Isoforms

Nitric oxide synthase (NOS) catalyzes the conversion of L -arginine, molecular oxygen, and nicotinamide adenine dinucleotide phosphate (NADPH) to NO, citrulline, and NADP⁺ (reviewed in ref. 1 ). The neuronal (n) and endothelial (e) NOS isozymes are highly regulated by Ca²⁺ and calmodulin (CaM), whereas the iNOS has CaM tightly bound. NOS are heme proteins that also contain tightly bound flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and require tetrahydrobiopterin (BH₄ ) for activity, whereas NADPH is used as a substrate.