Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts.
C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.
Wolff
D J
DJ
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA. wolffdj@umdnj.edu
Papoiu
A D
AD
Mialkowski
K
K
Richardson
C F
CF
Schuster
D I
DI
Wilson
S R
SR
D000818:Animals; D002244:Carbon / Q000737:chemistry* / Q000494:pharmacology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002956:Citrulline / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D037741:Fullerenes; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D008314:Malonates / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D009245:NADH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D009474:Neurons / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II; D010902:Pituitary Gland / Q000201:enzymology; D011487:Protein Conformation; D020033:Protein Isoforms; D051381:Rats; D013997:Time Factors
D037741:Fullerenes; D008314:Malonates; D020033:Protein Isoforms; D002956:Citrulline; D009569:Nitric Oxide; D002244:Carbon; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; D019255:NADPH Oxidases; D009245:NADH Dehydrogenase; C069837:fullerene C60