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

Jimmy R. Calaycay*, Theresa M. Kelly, Karen L. MacNaul, Ermenegilda D. McCauley, Hongbo Qi, Stephan K. Grant, Patrick R. Griffin, Tracey Klatt, S. M. Raju, Andreas K. Nussler, Shrenik Shah, Jeffrey R. Weidner, Hollis R. Williams, Gloria C. Wolfe, David A. Geller, Timothy R. Billiar, Malcolm MacCoss, Richard A. Mumford, Michael J. Tocci, John A. SchmidtKenny K. Wong, Nancy I. Hutchinson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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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 °C and contained 1.15 ± 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret λ(max) at 396 nm with a shoulder at 460 nm and contained 0.28-0.64 tool 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 (K(i) = 22.6 ± 1.9 nM), reversed the type II difference spectrum induced by imidazole (K(d) = 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.

Original languageEnglish
Pages (from-to)28212-28219
Number of pages8
JournalJournal of Biological Chemistry
Issue number45
StatePublished - 1996
Externally publishedYes


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