A(2A) adenosine receptors were examined in bovine striatal membranes following exposure to tetranitromethane (TNM) which modifies tyrosine and cysteine residues. TNM (0.05-0.5 mM) treatment caused an irreversible, concentration-dependent decrease in the binding activity of the selective A(2A) agonist [H-3]CGS 21680. Protection studies showed that TNM inactivation could be prevented by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) and by the antagonist xanthine amine congener (XAC), suggesting that TNM modified residues at the ligand-binding sites. Scatchard analysis of the binding data showed that 0.15 mM TNM decreased the [H-3]CGS 21680 B-max value from 447 +/- 39 to 273 +/- 21 fmol/mg of proteins without any significant change in the K-d values (13.5 +/- 1.4 and 14.7 +/- 1.5 for control and treated membranes, respectively). We carried out a series of successive chemical modifications with the reducing agent dithiothreitol (DTT), which indicated that the residues modified by TNM, under our experimental conditions, are tyrosine residues and not cysteine residues.
Chemical modifications of striatal A(2A) adenosine receptors: A possible role for tyrosine at the ligand binding sites
MARTINI, CLAUDIA;TRINCAVELLI, MARIA LETIZIA;LUCACCHINI, ANTONIO
1997-01-01
Abstract
A(2A) adenosine receptors were examined in bovine striatal membranes following exposure to tetranitromethane (TNM) which modifies tyrosine and cysteine residues. TNM (0.05-0.5 mM) treatment caused an irreversible, concentration-dependent decrease in the binding activity of the selective A(2A) agonist [H-3]CGS 21680. Protection studies showed that TNM inactivation could be prevented by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) and by the antagonist xanthine amine congener (XAC), suggesting that TNM modified residues at the ligand-binding sites. Scatchard analysis of the binding data showed that 0.15 mM TNM decreased the [H-3]CGS 21680 B-max value from 447 +/- 39 to 273 +/- 21 fmol/mg of proteins without any significant change in the K-d values (13.5 +/- 1.4 and 14.7 +/- 1.5 for control and treated membranes, respectively). We carried out a series of successive chemical modifications with the reducing agent dithiothreitol (DTT), which indicated that the residues modified by TNM, under our experimental conditions, are tyrosine residues and not cysteine residues.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
