Purification and high-sensitivity membrane photoaffinity labeling of mammalian beta/sub 2/-adrenergic receptor
The Beta/sub 2/-Adrenergic receptor (BAR) from guinea pig lung has been purified to near homogeneity. The purified BAR, detected by silver staining or by total radioiodination and autoradiography, migrates on SDS-PAGE as a broad band centered at 66 kilodaltons (kD). This band can be specifically labeled with the adrenergic photoaffinity ligand, /sup 125/I-azidobenzylpindolol. The purified BAR displays the same beta/sub 2/-subtype pharmacology and mobility on SDS-PAGE as the membrane-bound BAR. Microsequenator analysis of the purified BAR suggests that the amino terminus of the receptor is blocked. Several site-specific agents were used to fragment the purified BAR; some of the fragments may be useful for obtaining amino acid sequence of the BAR. Conditions also have developed for photoaffinity labeling the BAR in membranes of mammalian tissue culture cells (human astrocytoma, 1321N1) which contain very low levels of BAR. The BAR from these cells migrates as a broad band of about 66 kD on SDS-PAGE. Endoglycosidase F, which cleaves N-linked oligosaccharides, reduces the apparent molecular weight of the BAR from these cells to 45 kD. Recovery from agonist-induced down-regulation in post-confluent cultures of 1321N1 cells in the presence of tunicamycin (an inhibitor of N-linked glycosylation) results in the appearance of a 41 kD form of the BAR. Despite the apparent absence of N-linked oligosaccharides, this 41 kD form of the BAR retains adrenergic binding activity.
- Research Organization:
- North Carolina Univ., Chapel Hill (USA)
- OSTI ID:
- 6500193
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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LUNGS
AUTORADIOGRAPHY
RECEPTORS
PURIFICATION
SYMPATHOMIMETICS
RADIORECEPTOR ASSAY
CELL MEMBRANES
ELECTROPHORESIS
GUINEA PIGS
INDOLES
IODINATION
IODINE 125
OLIGOSACCHARIDES
TUMOR CELLS
ANIMAL CELLS
ANIMALS
AROMATICS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZAARENES
AZOLES
BETA DECAY RADIOISOTOPES
BODY
CARBOHYDRATES
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
HALOGENATION
HETEROCYCLIC COMPOUNDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PROTEINS
PYRROLES
RADIOISOTOPES
RESPIRATORY SYSTEM
RODENTS
SACCHARIDES
TRACER TECHNIQUES
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics