title: Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils.
abstract: Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.
authors.affiliation: Department of Internal Medicine, Faculty of Medicine, Kyoto University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Yui
authors.forename: R
authors.initials: R
authors.lastname: Hattori
authors.forename: K
authors.initials: K
authors.lastname: Kosuga
authors.forename: H
authors.initials: H
authors.lastname: Eizawa
authors.forename: K
authors.initials: K
authors.lastname: Hiki
authors.forename: S
authors.initials: S
authors.lastname: Ohkawa
authors.forename: K
authors.initials: K
authors.lastname: Ohnishi
authors.forename: S
authors.initials: S
authors.lastname: Terao
authors.forename: C
authors.initials: C
authors.lastname: Kawai
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D009504:Neutrophils / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D002147:Calmodulin; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases