Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.
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, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.
Stuehr
D J
DJ
Department of Medicine, Cornell University Medical College, New York, NY 10021.
Cho
H J
HJ
Kwon
N S
NS
Weise
M F
MF
Nathan
C F
CF
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D016207:Cytokines / Q000494:pharmacology*; D004790:Enzyme Induction; D004926:Escherichia coli; D005486:Flavin Mononucleotide / Q000032:analysis*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis*; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D019001:Nitric Oxide Synthase
D002147:Calmodulin; D016207:Cytokines; D008070:Lipopolysaccharides; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D002118:Calcium