PMID: 2500980Jun 26, 1989Paper

Reconstitution and characterization of the human neutrophil N-formyl peptide receptor and GTP binding proteins in phospholipid vesicles

Biochimica Et Biophysica Acta
J F CupoG M Bokoch

Abstract

We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.

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Citations

Jun 16, 2010·European Biophysics Journal : EBJ·Subhash Chandra YadavMedicherla V Jagannadham
Jan 1, 1991·The International Journal of Biochemistry·T Cserháti, M Szögyi
Aug 28, 2003·Life Sciences·Roland Seifert, Katharina Wenzel-Seifert
Feb 5, 2003·Biochemical and Biophysical Research Communications·Katharina Wenzel-Seifert, Roland Seifert
Sep 14, 2011·Journal of the American Chemical Society·Sophie RoizardHorst Vogel
May 6, 2006·The Journal of Biological Chemistry·Brian A BabbinAsma Nusrat
Apr 20, 2001·The Journal of Biological Chemistry·T A BennettL A Sklar
Sep 18, 2001·The Journal of Biological Chemistry·R Seifert, K Wenzel-Seifert
Nov 24, 1999·The Journal of Biological Chemistry·K Wenzel-SeifertR Seifert
Sep 3, 1998·The Journal of Biological Chemistry·K Wenzel-SeifertR Seifert

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