Redox forms of human placenta glutathione transferase.
Human placenta glutathione transferase (EC 2.5.1.18) pi undergoes an oxidative inactivation which leads to the formation of an inactive enzymatic form which is homogeneous in several chromatographic and electrophoretic conditions. This process is pH dependent, and it occurs at appreciable rate in alkaline conditions and in the presence of metal ions. Dithiothreitol treatment completely restores the active form. -SH titration data and electrophoretic studies performed both on the oxidized and reduced forms indicate that one intrachain disulfide is formed, probably between the two faster reacting cysteinyl groups of each subunit. By the use of a specific fluorescent thiol reagent the disulfide forming cysteines have been identified as the 47th and 101th residues. The disulfide formation causes changes in the tertiary structure of this transferase as appears by CD, UV, and fluorometric analyses; evidences are provided that one or both tryptophanyl residues of each subunit together with a number of tyrosyl residues are exposed to a more hydrophilic environment in the oxidized form. Moreover, electrophoretic data indicate that the subunit of the oxidized enzyme has an apparent molecular mass lower than that of the reduced transferase, thereby confirming structural differences between these forms.
Institute of Biochemical Sciences, University of Chieti G. D'Annunzio, Italy.
G
G
Ricci
G
G
Del Boccio
A
A
Pennelli
M
M
Lo Bello
R
R
Petruzzelli
A M
AM
Caccuri
D
D
Barra
G
G
Federici
D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D005982:Glutathione Transferase; D005978:Glutathione; D004229:Dithiothreitol