Solubilization and hydrodynamic characterization of guanine nucleotide sensitive vasoactive intestinal peptide-receptor complexes from rat intestine

Journal Article · Tue Feb 21 00:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00430a036· OSTI ID:5719178

Calvo, J R; Couvineau, A; Guijarro, J; Laburthe, M ^[1]

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1. INSERM, Villejuif (France)

The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM {sup 125}I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1{mu}M unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound {sup 125}I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. After cholera toxin catalyzed ({sup 32}P)ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of {sup 32}P radioactivity was coeluted with the 5.2-nm component. SDS-PAGE analysis revealed the presence in this peak of the M{sub r} 42,000 {alpha} subunit of the Gs protein. Sucrose density gradient ultracentrifugation of the 5.1- and 3.1-nm components isolated on Sephacryl S-300 showed that they displayed apparent sedimentation coefficients of 6.7 and 3.9 S, respectively. From these results, the molecular weight of both components was estimated to be 152,000 and 54,000, respectively. It was concluded that intestinal VIP-receptor complexes of M{sub r} 54,000 were solubilized in a major form of M{sub r} 152,000 containing a Gs protein that was sensitive to GTP regulation.

• OSTI ID: 5719178
• Journal Information: Biochemistry; (USA), Vol. 28:4; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RECEPTORS
MOLECULAR STRUCTURE
BIOCHEMICAL REACTION KINETICS
CELL MEMBRANES
CHROMATOGRAPHY
FILTRATION
GUANINE
INTESTINES
IODINE 125
NUCLEOTIDES
PEPTIDES
PHOSPHORUS 32
RATS
TOXINS
AMINES
ANIMALS
ANTIGENS
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
GASTROINTESTINAL TRACT
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MATERIALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
PROTEINS
PURINES
RADIOISOTOPES
REACTION KINETICS
RODENTS
SEPARATION PROCESSES
TOXIC MATERIALS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques