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Reconstitution of rat liver vasopressin receptors into phospholipid vesicles

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:5096785
; ; ;

Isolation and characterization of the hepatic vasopressin (V1) receptor has been hampered by the loss of vasopressin binding when the receptor is solubilized. An alternative to a soluble binding assay is reconstitution of solubilized receptors into phospholipid vesicles. They report here the initial reconstitution of solubilized vasopressin receptors. Rat liver microsomes were solubilized with 3% 3-((3-cholamido-propyl)dimethyl-ammonio)-1-propanesulfonate (CHAPS) in 20mM HEPES (pH 7.4). The solubilized protein was combined with an equal volume of 7% soybean phospholipids dissolved in 3% CHAPS. This solution was passed over a column of Sephadex G50 equilibrated with 30 mM HEPES, 5mM MgCl/sub 2/ (pH 7.4). Reconstituted vesicles containing the microsomal proteins were recovered in the void volume. Binding of (/sup 3/H)-Vasopressin to the reconstituted vesicles at pH 7.4 was 80% specific, saturable, reversible and required magnesium. Solubilization of microsomes in the presence of glycerol, prebound ligand, phospholipids or magnesium did not improve subsequent binding. This reconstitution procedure will allow examination of signal transduction mechanisms and may be used as a functional assay in isolating the receptor.

Research Organization:
Boston Univ. School of Medicine, MA
OSTI ID:
5096785
Report Number(s):
CONF-8606151-
Journal Information:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CELL CONSTITUENTS
DIGESTIVE SYSTEM
ESTERS
GLANDS
HORMONES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIGANDS
LIPIDS
LIVER
MAMMALS
MEMBRANE PROTEINS
MICROSOMES
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANOIDS
ORGANS
PEPTIDE HORMONES
PHOSPHOLIPIDS
PITUITARY HORMONES
PROTEINS
RATS
REACTION KINETICS
RECEPTORS
RODENTS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VASOPRESSIN
VERTEBRATES