Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein.
The synthesis of nitric oxide (.NO) from L-arginine has been demonstrated in a number of cell types and functions either as a cell signaling agent or as a key component of the cell-mediated immune response. Both constitutive and inducible activities have been described. Herein we report the purification of inducible .NO synthase (EC 1.14.23) from activated murine macrophages using a two-column procedure. Crude 100,000 x g supernatant was passed through a 2'-5'-ADP-Sepharose 4B affinity column followed by a DEAE-Bio-Gel A anion exchange column. The .NO synthase ran as a band of Mr = 130,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration experiments using a Superose 6 HR 10/30 column estimated the native molecular weight to be 260 +/- 30 kDa, indicating that the native enzyme exists as a dimer. Activity was dependent upon L-arginine (Km = 16 +/- 1 microM at 37 degrees C and pH 7.5) and NADPH. Both (6R)-tetrahydro-L-biopterin and FAD enhanced activity, whereas Mg2+ and FMN had no effect on activity. Fluorescence studies demonstrated the presence of one bound FAD and one bound FMN per subunit.
Hevel
J M
JM
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
White
K A
KA
Marletta
M A
MA
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005420:Flavoproteins / Q000737:chemistry; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D019001:Nitric Oxide Synthase
D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases