Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.
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.
Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, Massachusetts.
C R
CR
Lyons
G J
GJ
Orloff
J M
JM
Cunningham
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004790:Enzyme Induction; D005260:Female; D015870:Gene Expression; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000201:enzymology; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic; D014982:Xenopus laevis
D009838:Oligodeoxyribonucleotides; D004247:DNA; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases