Glutathione transferase from bovine placenta. Preparation, biochemical characterization, crystallization, and preliminary crystallographic analysis of a neutral class PI enzyme.
A method was developed to purify glutathione transferase from bovine placenta by affinity chromatography and fast protein liquid chromatography. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. The dimeric enzyme is composed of identical subunits with a molecular weight of 23,000; its isoelectric point is 6.9. In contrast to previously described isoenzymes of glutathione transferase, the protein we have purified exists in two forms, an active reduced form and a less active oxidized form. These can be reversibly transformed into each other but behave differently in sedimentation analysis, gel chromatography, and gel electrophoresis. These differences may reflect a change in the molecular shape of glutathione transferase. Chemical modification with iodoacetate, iodoacetamide (presumably of thiol groups), phenylglyoxal, and butadione (presumably of arginyl groups), and their inhibitory effects on the activity were investigated. From substrate specificity studies and N-terminal sequence analysis it is obvious that this glutathione transferase must belong to the isoenzyme class pi. The purified enzyme could be crystallized from 1.4 M ammonium sulfate solution, pH 8.0, in the presence of S-hexyl-glutathione. The crystals are tetragonal, with space group P4(1)2(1)2 or P4(3)2(1)2. The cell constants are a = b = 6.1 nm, c = 23.7 nm, alpha = beta = gamma = 90 degrees. The crystals diffract to 0.26-nm resolution and are suitable for x-ray structure analysis.
Schäffer
J
J
Max-Planck-Institut für Biochemie, Martinsried, West Germany.
Gallay
O
O
Ladenstein
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D005982:Glutathione Transferase