RESUMO
Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Marcadores de Afinidade/química , Ativação Enzimática/fisiologia , Radicais Livres/química , Ligantes , Micrococcus luteus/enzimologia , Peptídeos/química , Conformação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/ultraestruturaRESUMO
Photoaffinity labeling is a special type of chemical modification, where the label is activated by the action of light. This article presents the general principles and limitations of this technique, its application to the study of Micrococcus luteus ATPase and the use of photoaffinity crosslinking to probe the structure of this enzyme
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Marcadores de Afinidade/química , Ativação Enzimática/fisiologia , Radicais Livres/química , Ligantes , Micrococcus luteus/enzimologia , Peptídeos/química , Conformação Proteica , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/ultraestruturaRESUMO
Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade/metabolismo , Azidas/metabolismo , Receptores Nicotínicos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , TorpedoRESUMO
UV irradiation of the ATPase (CF1) from spinach chloroplasts in the presence of 3'-arylazido-beta-alanyl-8-azido ATP (8,3'-DiN3ATP) results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of alpha-beta cross-links. The results demonstrate an interfacial localization of the nucleotide binding sites on CF1.
