Affinity Labeling of Steroid Binding Sites

作者: Manik Ganguly , James C. Warren

DOI: 10.1016/S0021-9258(18)62177-5

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摘要: Abstract To extend the application of affinity labeling for characterization macromolecular steroid binding sites, we synthesized two cortisone derivatives (cortisone 21-iodoacetate and 21-iododeoxycortisone) studied their reactions with use 20β-hydroxysteroid dehydrogenase from Streptomyces hydrogenans in 0.05 m phosphate buffer, pH 7.0, as model protein. Both are capable reversible step at active site, both serve substrates. Cortisone inactivates enzyme a time-dependent irreversible manner, slows rate this inactivation, excess 2-mercaptoethanol stops it. Iodoacetic acid did not inactivate (with or without added cortisone), nor 21-iododeoxycortisone. Nevertheless, easily react sulfhydryl compounds. Radioactive was then 2-3H-iodoacetic acid. Inactivation by accompanied radiolabeling; neither seen alone. After inactivation hydrolysis, single major fraction radioactivity on paper chromatography. Amino analysis hydrolysate revealed radioactive peak (containing 86% total radioactivity) mobility identical standard 1,3-dicarboxymethyl histidine minor 5% 3-carboxymethyl histidine. These observations compatible mechanism whereby moiety delivers reagent group to site dehydrogenase, where it reacts residue that site. offered compounds which may possibly be used study certain sites high present receptor proteins target organs.

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