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

Abstract

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon y 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 NOj plus NOj per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 FM, 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 -250 kDa but could be dissociated into inactive monomers of -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.The free radical nitric oxide (NO) or a NO-releasing product is synthesized within mammalian immune, cardiovascular, and neural systems, where it functions as a signaling or cytotoxic molecule (for reviews, see refs. 1-3). The mammalian enzymes that generate NO are not completely characterized. Current evidence suggests that there are at least two forms. One is constitutively expressed, requires calcium ions and a calcium-binding protein such as calmodulin for its activation, and participates in signal transduction by generating NO in response to increased intracellular calcium levels, leading to activation of soluble guanylyl cyclase by NO (1, 4-7). The other form is expressed in cells only after several hours of exposure to cytokines like interferon y (IFN-y) and/or microbial products such as bacterial lipopolysaccharide (LPS) (8)(9)(10). Immunologically induced NO synthase participates in the destruction of microbial pathogens and tumor cells and contributes to shock associated with sepsis (2, 3, 11, 12). The inducible NO synthase appears to be antigenically distinct from constitutive NO synthase (13).Despite their differences in regulation and function, evidence suggests that the constitutive and induced NO synthases are catalytically similar. Both types utilize NADPH and, where it was tested, tetrahydrobiopterin as redox cofactors (4-7, 14, 15) and convert L-arginine to NO and L-citrulline with one atom of molecular oxygen being incorporated into L-citrulline (16). In cases where it was tested, both constitutive and inducible activities were enhanced by exogenous FAD (7, 17) and inhibited irreversibly by the flavoprotein inhibitor diphenyleneiodonium (18), suggesting that NO synthases may be flavoproteins. Howev...

Cytoskeleton‐dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells

“…Under physiological conditions, the endothelial lining of vessels contributes to the regulation of vascular tone via NO production by the calcium/calmodulin-dependent constitutive NO synthase (ecNOS) (Furchgott & Zawadzki, 1980;Ignarro et al, 1987;Palmer et al, 1988;Vallance et al, 1989;Rees et al, 1989;Bredt et al, 1991). There is increasing evidence, however, for a different mechanism of NO formation via the inducible and calcium-independent NO synthase (iNOS) in a variety of cells, including macrophages (Stuehr et al, 1991;Xie et al, 1992)) and vascular smooth muscle cells (Fleming et al, 1990;Beasley, 1990;Beasley et al, 1989; Busse & Mulsch, 1990). Such an endotoxin-and cytokine-inducible pathway synthesizing large amounts of NO and increasing cyclic GMP in the vasculature appears to be responsible for the hypotension, reduced responsiveness to vasoconstrictors and cardiovascular collapse associated with septic shock (Kilbourne & Belloni 1990;Kilbourne et al, 1991), as well as for the side effects of anti-tumour therapy with cytokines (Moritz et al, 1989;Hibbs et al, 1992).…”

Nitric oxide localized to spinal cords of mice with experimental allergic encephalomyelitis: an electron paramagnetic resonance study.

“…Recently, it has been shown that activated macrophages (as well as other cell types) synthesize large amounts of NO via a distinct, inducible isoform of NOS (iNOS) (10). NO thus generated appears to be under immunologic control (11,12) and to be cytotoxic (13).…”

Endotoxin Decreases the Contractile Responses of the Porcine Basilar Artery to Vasoactive Substances

“…NO, which is synthesized from the terminal guanidino nitrogen atoms of the basic amino acid L-arginine (Palmer et ai., 1988) by NO synthases (Bredt and Snyder, 1990;Stuehr et al, 1991), induces vasodilation via activation of soluble guanylate cyclase (Ignarro, 1989;Moncada et aI., 1991). Accordingly, an over production of NO resulting from induction of an inducible NO synthase in endothelial and smooth muscle cells by endotoxin, and therefore a decrease in vascular reactivity, have been implicated in sep tic shock following bacterial infection (Knowles et aI., 1990;Radomski et aI., 1990;Salvemini et aI., 1990Salvemini et aI., , 1992.…”