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

J Biol Chem. 1992 Mar 25;267(9):6370-4.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Oxidoreductases / biosynthesis
  • Amino Acid Oxidoreductases / genetics*
  • Amino Acid Oxidoreductases / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • Cloning, Molecular / methods
  • DNA / genetics
  • DNA / isolation & purification
  • Enzyme Induction
  • Female
  • Gene Expression
  • Macrophages / enzymology
  • Mice
  • Molecular Sequence Data
  • Nitric Oxide Synthase
  • Oligodeoxyribonucleotides
  • Oocytes / enzymology
  • Polymerase Chain Reaction
  • Transcription, Genetic
  • Xenopus laevis


  • Oligodeoxyribonucleotides
  • DNA
  • Nitric Oxide Synthase
  • Amino Acid Oxidoreductases

Associated data

  • GENBANK/L01675
  • GENBANK/L01676
  • GENBANK/L01677
  • GENBANK/M81836
  • GENBANK/M83679
  • GENBANK/M83680
  • GENBANK/M83681
  • GENBANK/M83724
  • GENBANK/M83821
  • GENBANK/M84373