Purification of nitric oxide synthase from rat macrophages.
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'-diphosphate-agarose 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 Mr = 150,000. The purified enzyme is colorless, and an absorption maximum is observed at 280 nm. The half-life of the enzyme activity is 6 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of NADPH, (6R)-5,6,7,8-tetrahydro-L-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).
Yui
Y
Y
Department of Internal Medicine, Faculty of Medicine, Kyoto University, Japan.
Hattori
R
R
Kosuga
K
K
Eizawa
H
H
Hiki
K
K
Kawai
C
C
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase; D051381:Rats; D011919:Rats, Inbred Strains
D001708:Biopterins; D009249:NADP; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin; D004229:Dithiothreitol