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

C. R. Lyons, G. J. Orloff, J. M. Cunningham

Research output: Contribution to journalArticlepeer-review

Abstract

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-γ and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO·. Nitrite production is blocked by the enzyme inhibitor, N(G)- 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 Snyder, 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.

Original languageEnglish (US)
Pages (from-to)6370-6374
Number of pages5
JournalJournal of Biological Chemistry
Volume267
Issue number9
StatePublished - 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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