Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine

• Published in: The Journal of biological chemistry 1996-11, Vol.271 (45), p.28212-28219
• Main Authors: Calaycay, J R, Kelly, T M, MacNaul, K L, McCauley, E D, Qi, H, Grant, S K, Griffin, P R, Klatt, T, Raju, S M, Nussler, A K, Shah, S, Weidner, J R, Williams, H R, Wolfe, G C, Geller, D A, Billiar, T R, MacCoss, M, Mumford, R A, Tocci, M J, Schmidt, J A, Wong, K K, Hutchinson, N I
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid; Enzyme Induction; Humans; Kinetics; NADP - metabolism; Nitric Oxide Synthase - metabolism; Radiation-Protective Agents - pharmacology; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thiazines - pharmacology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.45.28212

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.