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

Academic Article
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Overview

authors

  • Calaycay, J. R.
  • Kelly, T. M.
  • MacNaul, K. L.
  • McCauley, E. D.
  • Qi, H. B.
  • Grant, S. K.
  • Griffin, Patrick
  • 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.

publication date

  • 1996

journal

  • Journal of Biological Chemistry  Journal

abstract

  • 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.

subject areas

  • Chromatography, High Pressure Liquid
  • Enzyme Induction
  • Humans
  • Kinetics
  • NADP
  • Nitric Oxide Synthase
  • Radiation-Protective Agents
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Thiazines
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 8910438
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Additional Document Info

start page

  • 28212

end page

  • 28219

volume

  • 271

issue

  • 45

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