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Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00414a062· OSTI ID:7188228
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The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity (/sup 3/H)fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity (/sup 3/H)fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

Research Organization:
Boston Univ. School of Medicine, MA (USA)
OSTI ID:
7188228
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:14; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
AMINO ACIDS
ANIMALS
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
BRAIN
CARBOXYLIC ACIDS
CATTLE
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CHEMISTRY
CONFIGURATION INTERACTION
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
EVEN-ODD NUCLEI
GUANINE
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPES
LABELLED COMPOUNDS
LEUCINE
LEUKOCYTES
LIGHT NUCLEI
MAMMALS
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR STRUCTURE
NERVOUS SYSTEM
NEUTROPHILS
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PHENYLALANINE
PROTEINS
PURINES
RABBITS
RADIOISOTOPES
RECEPTORS
RUMINANTS
SULFUR 35
SULFUR ISOTOPES
TRITIUM COMPOUNDS
VERTEBRATES