Chemical modification of histidine residues in rabbit liver glutathione reductase.
The role of histidine residues of glutathione reductase from rabbit liver was investigated by chemical modification with both ethoxyformic anhydride and dansyl chloride. At least four histidine residues were concomitantly modified by ethoxyformic anhydride at pH 6; both the GSSG reductase and the transhydrogenase activities were inhibited to the same extent. Dansyl chloride inactivated the enzyme showing pH-independence in the range 7-9. About 2.6 moles dansyl were incorporated in the protein 80% inactivated at pH 8, whereas at pH 7 a lower amount of labelling was found. Nearly complete reactivation of the inactivated enzyme could be obtained by incubation with hydroxylamine, which released all the acid-labile bound dansyl. Of the two histidine residues modified, only the slower reacting residue seems essential for activity. The modification with dansyl chloride will allow the identification of the histidine residues modified, in the sequence of the protein.
D000818:Animals; D003619:Dansyl Compounds / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005980:Glutathione Reductase / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D003619:Dansyl Compounds; D006898:Hydroxylamines; D019811:Hydroxylamine; D006639:Histidine; D005980:Glutathione Reductase; D004047:Diethyl Pyrocarbonate; C029038:dansyl chloride