Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet α2-adrenoceptors with TNM resulted in an irreversible loss of binding activity. Typically, an 80-90% decrease in binding activity occurred with a 60-min exposure to 320 μM TNM. The loss of α2-adrenoceptor activity caused by TNM could be prevented if α2-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by α2-adrenergic ligands was does-dependent and showed a positive correlation with the affinity of the ligand for the α2-adrenoceptor. Prazosin, an α1-specific antagonist, and propranolol, a β-adrenergic antagonist, did not protect α2-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in α2-adrenoceptor activity caused by TNM was due to a decrease in Bmax with no change in Kd. α2-Adrenoceptors were also inactivated with the sulfhydryl-specific reagent phenylmercuric chloride (PMC). The receptor inactivation caused by PMC could be reversed completely by subsequent treatment with dithiothreitol. Treatment of α2-adrenoceptors with combinations of TNM and PMC showed that the receptor inactivation caused by TNM was most likely due to an interaction with tyrosine residues. These results indicate that tyrosine residues have a function in the conformational stability of α2-adrenoceptors and may be directly involved with ligand binding to the receptor.
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