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Purification of nitric oxide synthase from rat macrophages

1991; Elsevier BV; Volume: 266; Issue: 19 Linguagem: Inglês

10.1016/s0021-9258(18)98932-5

1083-351X

Yoshiki Yui, Ryuichi Hattori, K Kosuga, Hiroshi Eizawa, Kazuaki Hiki, C Kawai,

Adipose Tissue and Metabolism

Nitric oxide (NO) synthase (EC 1.14.23) has been purified to apparent homogeneity from rat macrophages.The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphateagarose and gel filtration chromatography on a Superose 12 HR 10/30 column.The apparent molecular weight is 300,000 by gel filtration.On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrates as a single protein band with M, = 150,000.The purified enzyme is colorless, and an absorption maximum is observed at 280 nm.The halflife of the enzyme activity is 6 h at pH 7.4 and 4 "C.The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-~-biopterin, and dithiothreitol.Although the cerebellar and endothelial enzyme require Ca2+ and calmodulin, these are not required by the macrophage enzyme.The macrophage nitric oxide synthase (an inducible enzyme) seems to be different from the cerebellar and endothelial enzyme (a constitutive enzyme).Nitric oxide is synthesized in macrophages (l), neutrophils (2), endothelial cells (3), cerebellum (4), and hepatocytes (5).Using partially purified macrophage nitric oxide synthase, the reaction mechanism and characteristics of this enzyme have been studied Nitric oxide is synthesized from a guanidino nitrogen of L-arginine (6).NADPH is absolutely necessary for the reaction (7-10).(6R)-BHd1 is also required as a cofactor (8, 9).FAD and thiol are necessary for maximal nitric oxide generation (7).The oxygen atom in the ureido group of L-citrulline originates from dioxygen, not water (10).Recently, nitric oxide synthase was purified to near homogeneity from cerebellum, and the enzyme activity was found t o be dependent on Ca2+ and calmodulin (4, 11).Until now, however, the macrophage enzyme has not been purified to homogeneity.In this study, macrophage nitric oxide synthase was purified to the homogeneity, and some of its characteristics were investigated.