Work

Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein.

Year: 1991

Type: article

Abstract: 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.

Source: Journal of Biological Chemistry

DOI: 10.1016/s0021-9258(18)54421-5

Authors: J.M. Hevel, K.A. White, M.A. Marletta

Institution: University of Michigan

Language: English

Topic: Nitric Oxide and Endothelin Effects

Subfield: Physiology (medical)

Field: Medicine

Domain: Health Sciences

Open Access status: hybrid

Estimated APC paid: $3,430

Funder: National Cancer Institute