Photoaffinity labeling of A1-adenosine receptors

Journal Article · Mon Nov 25 04:00:00 EST 1985 · J. Biol. Chem.; (United States)

OSTI ID:6113921

Klotz, K N; Cristalli, G; Grifantini, M; Vittori, S; Lohse, M J

The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for (TH)N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of (TH)N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity ( SVI-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for SVI-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that SVI-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.

• OSTI ID: 6113921
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 27; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

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59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ADENOSINE
ADIPOSE TISSUE
AFFINITY
ANIMAL TISSUES
ANIMALS
AZIDES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
BIOLOGICAL MATERIALS
BIOLOGICAL RADIATION EFFECTS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY
BODY FLUIDS
BRAIN
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CONNECTIVE TISSUE
COVALENCE
CYCLASES
ELECTROMAGNETIC RADIATION
ELECTROPHORESIS
ENZYMES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LABELLING
LIGANDS
LYASES
MAMMALS
MATERIALS
MEMBRANES
NERVOUS SYSTEM
NITROGEN COMPOUNDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANS
PEPTIDES
PROTEINS
RADIATION EFFECTS
RADIATIONS
RATS
REACTION KINETICS
RECEPTORS
RIBOSIDES
RODENTS
TISSUES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
VERTEBRATES