Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein

摘要: A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, gel filtration chromatography. SDS/PAGE of the NO gave three closely spaced silver-staining protein bands between 125 135 kDa. When assayed in presence L-arginine, NADPH, tetrahydrobiopterin, FAD, reduced thiol, had specific 1313 nmol NO2- plus NO3- per min mg. The apparent Km enzyme for L-arginine NADPH 2.8 0.3 microM, respectively. Addition calcium ions or without calmodulin did not increase enzyme, synthesis altered inhibitors. Gel chromatography indicated that induced catalytically competent as dimer approximately 250 kDa but could be dissociated into inactive monomers 130 absence tetrahydrobiopterin. Upon heat denaturation, released 1.1 mol FAD 0.55 FMN 130-kDa subunit. Thus, inducible differs several respects constitutive synthases is one very few eukaryotic enzymes containing both FMN.