TY - JOUR
T1 - Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree
AU - Myatt, Leslie
AU - Brockman, Diane E.
AU - Langdon, Gretchen
AU - Pollock, Jennifer S.
PY - 1993
Y1 - 1993
N2 - We have characterized the NO synthase enzyme in the villous vasculature of the human placenta as part of our ongoing studies of the regulation of NO synthesis in this circulation. NO synthase activity was determined by conversion of 3H l-arginine to 3H l-citrulline in cellular homogenate, cytosolic and particulate fractions. Optimal NO synthase activity was measured in all fractions in the presence of 1 mm NADPH, 10 μm tetrahydrobiopterin, 2 μm FAD, 100 μm free calcium and 50 U/ml calmodulin. The calmodulin inhibitor calmidazolium (50 μm) and FAD inhibitor diphenyliodonium chloride (1 μm) significantly reduced enzyme activity. The EC50 for calcium was 0.1 μm and Km for l-arginine 2.00±0.49 μm with Vmax 55.8±28.3 pmoles/mg protein/min. Enzyme activity was inhibited in both cytosolic and particulate fractions by ng-nitro-l-arginine and ng-monomethyl-l-arginine in a concentration-dependent manner (10−8–10−4 m). A calcium-independent NO synthase activity was also determined, but only constituted between 5–6 per cent of total activity. On Western blotting, a single 135 kda species was identified in each fraction with a monoclonal antibody raised against bovine aortic endothelial NO synthase. The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform.
AB - We have characterized the NO synthase enzyme in the villous vasculature of the human placenta as part of our ongoing studies of the regulation of NO synthesis in this circulation. NO synthase activity was determined by conversion of 3H l-arginine to 3H l-citrulline in cellular homogenate, cytosolic and particulate fractions. Optimal NO synthase activity was measured in all fractions in the presence of 1 mm NADPH, 10 μm tetrahydrobiopterin, 2 μm FAD, 100 μm free calcium and 50 U/ml calmodulin. The calmodulin inhibitor calmidazolium (50 μm) and FAD inhibitor diphenyliodonium chloride (1 μm) significantly reduced enzyme activity. The EC50 for calcium was 0.1 μm and Km for l-arginine 2.00±0.49 μm with Vmax 55.8±28.3 pmoles/mg protein/min. Enzyme activity was inhibited in both cytosolic and particulate fractions by ng-nitro-l-arginine and ng-monomethyl-l-arginine in a concentration-dependent manner (10−8–10−4 m). A calcium-independent NO synthase activity was also determined, but only constituted between 5–6 per cent of total activity. On Western blotting, a single 135 kda species was identified in each fraction with a monoclonal antibody raised against bovine aortic endothelial NO synthase. The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform.
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U2 - 10.1016/S0143-4004(05)80459-X
DO - 10.1016/S0143-4004(05)80459-X
M3 - Article
C2 - 7504255
AN - SCOPUS:0027301877
SN - 0143-4004
VL - 14
SP - 373
EP - 383
JO - Placenta
JF - Placenta
IS - 4
ER -