Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes
An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of (125I) human FSH ((125I) hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of (125I)hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of (125I)hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of (125I) hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with (3H)Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol (3H)Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of (3H)Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system.
- Research Organization:
- Albany Medical College, NY (USA)
- OSTI ID:
- 6681922
- Journal Information:
- Mol. Endocrinol.; (United States), Vol. 2:5
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
FSH
RECEPTORS
BIOCHEMICAL REACTION KINETICS
CELL MEMBRANES
CHOLESTEROL
DETERGENTS
IODINE 125
LIPOSOMES
PHOSPHOLIPIDS
POLYETHYLENE GLYCOLS
TEMPERATURE DEPENDENCE
TIME DEPENDENCE
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ADDITIVES
ALCOHOLS
BETA DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
EMULSIFIERS
ESTERS
GLYCOLS
GONADOTROPINS
HORMONES
HYDROXY COMPOUNDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIPIDS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
ORGANIC POLYMERS
ORGANOIDS
PEPTIDE HORMONES
PITUITARY HORMONES
POLYMERS
PROTEINS
RADIOISOTOPES
REACTION KINETICS
STEROIDS
STEROLS
SURFACTANTS
WETTING AGENTS
550201* - Biochemistry- Tracer Techniques