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tion and 2′,5′-ADP-sepharose affinity chromatography and
eluted with excess 2′-adenosine monophosphate (AMP) as
previously described.49 To exclude the possibility that the
purification procedure per se influenced the inhibitory profile
of the anti-pterins, native NOS-I was additionally purified by
diethyl aminoethyl (DEAE) ion exchange chromatography (200
mM, NaCl elution buffer; 20-25 mL min-1, flow rate) instead
of ammonium sulfate precipitation and subsequent 2′,5′-ADP-
sepharose affinity chromatography. Finally, the enzyme was
further purified in the presence of Ca2+ using CaM-sepharose
4 B and eluted by chelating Ca2+ with excess EGTA.7 The yield
of this purification method was 0.50 mg of enzyme from 1.00
kg of tissue with greater than 90% purity and a specific activity
of up to 670 nmol L-citrulline mg-1 min-1. Protein concentra-
tions were determined spectrophotometrically according to a
modified Bradford method50 using bovine serum albumin as a
standard and a Microplate reader (Molecular Devices; Sunny-
vale, CA). The purity was established from densitometric
scanning of Coomassie-stained SDS-PAGE gels using NIH
Image software (National Institutes of Health; Bethesda, MD).
NOS-I immunoreactive bands were examined by Western blot
analysis using an ECL enhanced chemiluminescense kit
(Amersham; Braunschweig, Germany) and a NOS-I specific
antibody (Transduction Laboratories; Hamburg, Germany).
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En zym e Activity. NOS-I enzyme activity was determined
from the calcium/calmodulin-dependent conversion of [3H]L-
arginine to [3H]L-citrulline.3,49 NOS-I was incubated for 15 min
at pH ) 7.2 and 37 °C in a mixture containing 2 µM H4Bip,
50 nM CaM, 1 mM CaCl2, 5 µM FAD, 10 µM FMN, 250 µM
3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-pro-
panesulfonat (Chapso), 50 mM triethanolamine (TEA), 1 mM
NADPH, 7 mM GSH, and 50 µM L-arginine + [2,3,4,5-3H]L-
arginine, and the reaction was stopped by adding ice-cold
acetate buffer (pH ) 5.5). [3H]L-Citrulline was separated from
the mixture by cation exchange chromatography, and the
amount of radioactivity was determined by liquid scintillation
counting. The effect of the anti-pterins, at an initial inhibitor
concentration of 100 µM, on NOS-I stimulated total activity
in the presence of 2 µM H4Bip was examined. Dose-response
curves of the most effective inhibitors (>50% inhibition of
stimulated NOS activity) in the range from 0 to 1000 µM were
constructed, and the corresponding IC50 values were deter-
mined by nonlinear regression analysis (UltraFit software
Biosoft, Cambridge, U.K., or GraphPad software, Prism,
Version 2.0a, San Diego, CA).
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macrophage function. Annu. Rev. Immunol. 1997, 15, 323-350.
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target for new drugs. Med. Res. Rev. 1994, 14, 23.
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L.; Marta´sek, P. Neuronal nitric oxide synthase, a modular
enzyme formed by convergent evolution: structure studies of a
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(15) (a) Kwon, N. S.; Nathan, C. F.; Stuehr, D. J . Reduced biopterin
as a cofactor in the generation of nitrogen oxides by murine
macrophages. J . Biol. Chem. 1989, 264, 20496-20501. (b) Tayeh,
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a cofactor. J . Biol. Chem. 1989, 264, 19654-19658. (c)
Schmidt, H. H. H. W.; Smith, R. M.; Nakane, M.; Murad, F. Ca2+
/
Ack n ow led gm en t. This work was supported by the
Bundesministerium fu¨r Bildung, Wissenschaft, Fors-
chung und Technologie (BMBF), and Deutsche Fors-
chungsgemeinschaft (SFB 355/C7). Peter Kotsonis is a
recipient of a C.J . Martin fellowship (Australian
NH&MRC) and the I2KF Wu¨rzburg. We thank Mr.
Manfred Bernhardt and Mrs. Birgit Thur for excellent
technical assistance.
calmodulin-dependent NO synthase type I: a biopteroflavopro-
tein with Ca2+/calmodulin-independent diaphorase and reduc-
tase activities. Biochemistry 1992, 31, 3243-3249. (d) Reif, A.;
Fro¨hlich, L. G.; Kotsonis, P.; Frey, A.; Bo¨mmel, H. M.; Wink, D.
A.; Pfleiderer, W.; Schmidt, H. H. H. W. Tetrahydrobiopterin
inhibits monomerization and is consumed during catalysis in
neuronal NO synthase. J . Biol. Chem. 1999, 274, 24921-24929.
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synthase reveals a role for tetrahydrobiopterin and L-arginine
in the formation of an SDS-resistant dimer. EMBO J . 1995, 14,
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