Purification of a distinctive form of endotoxin-induced nitric oxide synthase from rat liver.
An endotoxin-induced form of nitric oxide synthase (EC 1.14.23) was purified to homogeneity from rat liver by sequential anion-exchange chromatography and affinity chromatography using 2',5'-ADP-Sepharose. The enzyme has a subunit molecular mass of 135 kDa as determined by SDS/PAGE, a maximum specific activity of 462 nmol of citrulline formed from arginine per min per mg, and a Km for arginine of 11 microM. The enzyme was strongly stimulated by the addition of calmodulin with an EC50 of 2 nM, but removal of free calcium from the assay medium only reduced activity by 15%. Calmodulin inhibitors significantly reduced the enzyme activity. Tetrahydrobiopterin, FAD, and FMN were all required for full enzyme activity. This form of endotoxin-induced nitric oxide synthase from liver differs from the inducible enzyme found in macrophages and is unusual in that it is stimulated by calmodulin with little dependence on the calcium ion concentration.
Evans
T
T
Department of Infectious Diseases, Royal Postgraduate Medical School, London, United Kingdom.
Carpenter
A
A
Cohen
J
J
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004731:Endotoxins / Q000494:pharmacology*; D004790:Enzyme Induction; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D007711:Klebsiella pneumoniae; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D019001:Nitric Oxide Synthase; D051381:Rats; D011919:Rats, Inbred Strains
D002147:Calmodulin; D004731:Endotoxins; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D001708:Biopterins; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin