ISOLATION OF NITRIC-OXIDE SYNTHASE FROM HUMAN PLATELETS

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 100000 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 (similar to 80 kDa) under denaturing condition (SDS-PAGE). The native enzyme appears to be dimeric, since its molecular weight estimated by gel filtration was similar to 150 kDa. Enzyme activity was dependent on L-arginine, NADPH and (6R)-5,6,7,8-tetrahydro-L-biopterine. Partially purified platelet NOS (100000 X g supernatant) activity was sensitive to calmodulin antagonists and to the N-omega-Monomethyl-L-arginine, a substrate analog of L-arginine.