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Physicochemical properties of the N-formyl peptide receptor on human neutrophils

Academic Article
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Overview

authors

  • Allen, R. A.
  • Jesaitis, A. J.
  • Sklar, L. A.
  • Cochrane, Charles
  • Painter, R. G.

publication date

  • February 1986

journal

  • Journal of Biological Chemistry  Journal

abstract

  • In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.

subject areas

  • Affinity Labels
  • Azides
  • Chromatography, Gel
  • Detergents
  • Glycoproteins
  • Humans
  • Neutrophils
  • Oligopeptides
  • Receptors, Formyl Peptide
  • Receptors, Immunologic
  • Solubility
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 3944114
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Additional Document Info

start page

  • 1854

end page

  • 1857

volume

  • 261

issue

  • 4

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