Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.
Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.
Calaycay
J R
JR
Department of Molecular Design, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, USA. jimmy_calaycay@merck.com
Kelly
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MacNaul
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McCauley
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Qi
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Grant
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Griffin
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Klatt
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Raju
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Nussler
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Shah
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Weidner
J R
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Williams
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Wolfe
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Geller
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Billiar
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MacCoss
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Mumford
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Tocci
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Schmidt
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Wong
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Hutchinson
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D002851:Chromatography, High Pressure Liquid; D004790:Enzyme Induction; D006801:Humans; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013843:Thiazines / Q000494:pharmacology*
D011837:Radiation-Protective Agents; D013843:Thiazines; C018450:2-amino-5,6-dihydro-4H-1,3-thiazine; D009249:NADP; D019001:Nitric Oxide Synthase