Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line

• Published in: The Journal of biological chemistry Vol. 267; no. 9; pp. 6370 - 6374
• Main Authors: LYONS, C. R, ORLOFF, G. J, CUNNINGHAM, J. M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.03.1992
• Subjects: Amino Acid Oxidoreductases - biosynthesis; Amino Acid Oxidoreductases - genetics; Amino Acid Oxidoreductases - metabolism; Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; cDNA; Cell Line; Cloning, Molecular - methods; DNA - genetics; DNA - isolation & purification; Enzyme Induction; expression; Female; function; Fundamental and applied biological sciences. Psychology; Gene Expression; genes; Macrophages - enzymology; Mice; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nitric Oxide Synthase; nucleotide sequence; Oligodeoxyribonucleotides; Oocytes - enzymology; Polymerase Chain Reaction; predictions; Transcription, Genetic; Xenopus laevis; Nucleotide sequence; Enzyme; Induction; Cytokine; Rodentia; Vertebrata; Mammalia; Mouse; Lipopolysaccharide; Molecular cloning; Aminoacid sequence; Gamma interferon; Macrophage
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)42704-4

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.