Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart

Journal Article · Fri Apr 25 04:00:00 EST 1986 · J. Biol. Chem.; (United States)

OSTI ID:5620923

Terman, B I; Insel, P A

The photoaffinity probe (125I)aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited (3H)prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of (3H)prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on (3H)prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. The DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. Results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites.

• Research Organization: Univ. of California, San Diego, La Jolla
• OSTI ID: 5620923
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 12; ISSN JBCHA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AUTORADIOGRAPHY
AZIDES
BETA DECAY RADIOISOTOPES
BODY
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
HEART
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLED COMPOUNDS
LABELLING
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MUSCLES
MYOCARDIUM
NITROGEN COMPOUNDS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDES
PHOTOCHEMICAL REACTIONS
POLYPEPTIDES
PROTEINS
RADIOISOTOPES
RATS
RECEPTORS
RODENTS
TRITIUM COMPOUNDS
VERTEBRATES