title: Isolation of nitric oxide synthase from human platelets.
abstract: We are reporting a distinct constitutive isoform of nitric oxide synthase that has been purified to homogeneity from human platelet cytosolic fractions. Purification involved ultra centrifugation at 100,000 x g followed by two sequential affinity chromatography procedures: adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin Sepharose 4B. Purified enzyme appeared as a single band (approximately 80 kDa) under denaturing condition (SDS-PAGE). The native enzyme appears to be dimeric, since its molecular weight estimated by gel filtration was approximately 150 kDa. Enzyme activity was dependent on L-arginine, NADPH and (6R)-5,6,7,8-tetrahydro-L-biopterine. Partially purified platelet NOS (100,000 x g supernatant) activity was sensitive to calmodulin antagonists and to the N omega-Monomethyl-L-arginine, a substrate analog of L-arginine.
authors.affiliation: Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Muruganandam
authors.forename: B
authors.initials: B
authors.lastname: Mutus
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D001792:Blood Platelets / Q000201:enzymology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D019001:Nitric Oxide Synthase; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D013449:Sulfonamides; C017967:W 7; C034700:N-(6-aminohexyl)-1-naphthalenesulfonamide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases