Calmodulin is a subunit of nitric oxide synthase from macrophages.

Abstract

SummaryA central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist-induced elevation of Ca 2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca 2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino add sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca ~+ . This may explain why NOS that is independent of Ca 2+ and elevated CaM appears to be activated simply by being synthesized.

Molecular cloning of a cDNA encoding an inducible calmodulin‐dependent nitric‐oxide synthase from rat liver and its expression in COS 1 cells

“…This calmodulin independence was confirmed by the molecular cloning and sequence determination of murine macrophage NO synthase cDNA [8]. However, more recent studies on the structure of murine macrophage NO synthase cDNA suggests the presence of a putative calmodulin-binding site for the enzyme [lo] and this is consistent with the finding that calmodulin could be a subunit of the macrophage NO synthase [13]. We also have identified and purified an inducible calmodulindependent NO synthase in the liver of rats treated with Prupionibncterium acnes and Escherichia coli lipopolysaccharide (LPS) [14, 151. To obtain more information on the biological roles of inducible NO synthase(s) and the moleciilar basis of control of their enzymic activity and gene expression, we have cloned a cDNA for the inducible N O synthasc from rat liver.…”

“…Recently, Cho et al [13] found that calmodulin-dependent NO synthase is also strongly induced in murine macrophage-like cell lines. RAW 264.7, by treatment with LPS.…”

Inflammatory Modulation of Hepatocyte Apoptosis by Nitric Oxide: In Vivo, In Vitro, and In Silico Studies

“…This isoform is not typically expressed in the cellular resting state (exceptions include intestinal and bronchial epithelial cells, and renal tubular epithelial cells. [6,7] Instead, gene transcription is activated under acute inflammatory or stress conditions, and because calmodulin binds to iNOS in the absence of calcium elevations, [10] the enzyme produces NO in large quantities in a sustained manner. Pro-inflammatory cytokines, microbial products, and oxidative stress are all particularly effective at stimulating iNOS transcription.…”

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

“…The N-terminal oxygenase domain of NOS contains one mole equivalent of haem and possesses binding sites for L-arginine and (6R)-5,6,7,8-tetrahydrobiopterin (BH % [12]). The reductase and oxygenase domains are linked by a calmodulin (CaM) binding sequence [8,13,14] ; CaM binding facilitates electron transfer from the reductase to the oxygenase domain [15] and is proposed also to enhance the rate of interflavin electron transfer [16][17][18]. Of the three NOS isoforms, the inducible NOS (iNOS) isoform is expressed with CaM tightly bound [13] and regulation of activity is primarily through transcriptional processes ; the activities of endothelial NOS (eNOS) and neuronal NOS (nNOS) are regulated by CaM Abbreviations used : BH 4 , (6R)-5,6,7,8-tetrahydro-L-biopterin ; CaM, calmodulin ; CPR, cytochrome P450 reductase ; NOS, nitric oxide synthase ; eNOS, endothelial NOS ; FAD hq , hydroquinone FAD ; FAD sq semiquinone FAD ; FMN ox , oxidized FMN ; FMN sq , semiquinone FMN ; iNOS, inducible NOS ; nNOS, neuronal NOS ; nNOS CaM + , nNOS reductase complexed with CaM ; nNOS CaM − , nNOS reductase in which CaM has been removed by treatment with EDTA.…”