Photoaffinity labeling of the follitropin receptor
A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The SVI-labeled hormone derivative associated with the same number of receptors as SVI-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the SVI-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the SVI-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the SVI-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.
- Research Organization:
- Univ. of Wyoming, Laramie
- OSTI ID:
- 6086520
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 26; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
AZIDES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHEMISTRY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
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GONADOTROPINS
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IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
MAMMALS
MEMBRANES
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HORMONES
PHOTOCHEMICAL REACTIONS
PHOTOCHEMISTRY
PHOTOLYSIS
PITUITARY HORMONES
POLYMERIZATION
RADIOISOTOPES
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RECEPTORS
SWINE
TRACER TECHNIQUES
VERTEBRATES