Enhanced expression, purification, and characterization of a novel class alpha glutathione S-transferase isozyme appearing in rabbit hepatic cytosol following treatment with 4-picoline.
A novel class alpha glutathione S-transferase (GST) isozyme is expressed in the hepatic cytosol of rabbits treated with 4-picoline. SDS-PAGE analysis revealed the presence of a new 28-kDa band which cross-reacted with class alpha GST-specific IgG. This new GST isozyme was isolated from the hepatic cytosol of 4-picoline-treated rabbits and purified to homogeneity using S-hexylglutathione-agarose, CM-Sepharose, and PBE118 chromatofocusing chromatography. The isozyme was determined by SDS-PAGE and gel filtration analyses to be a homodimer of approximately 28 kDa with blocked N-terminus. A heterodimer consisting of 25 and 28 kDa subunits with activity toward the substrate 1-chloro-2,4-dinitrobenzene was also purified. Immunoblot analysis revealed that the 25, 26.5, and 28 kDa bands cross-reacted with class alpha GST-specific IgG and failed to react with either class mu or class pi GST-specific antibodies. The 28 kDa enzyme had a pI of 8.2 as determined by nonequilibrium pH gel electrophoresis. The purified 28 kDa enzyme exhibited activity toward 1-chloro-2,4-dinitrobenzene (Km = 1.60 mM and Vmax = 73.5 mumol/min/mg) and cumene hydroperoxide (Km = 1.02 mM and Vmax = 6.92 mumol/min/mg). Amino acid sequence analysis of several fragments resulting from cyanogen bromide cleavage of the 28 kDa GST isozyme revealed a class alpha GST consensus sequence. In addition, proteolytic digestion with alpha-chymotrypsin yielded peptide maps which showed distinct differences between the purified 28 kDa GST and another purified class alpha GST isozyme present in rabbit liver. These results provide evidence that class alpha GST isozymes containing a novel 28 kDa subunit are expressed following treatment with 4-picoline.
Primiano
T
T
Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201.
Novak
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D003488:Cyanogen Bromide / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000031:analogs & derivatives; D005982:Glutathione Transferase / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D010847:Picolines / Q000494:pharmacology*; D011817:Rabbits; D012685:Sepharose; D012689:Sequence Homology, Nucleic Acid
D007527:Isoenzymes; D010847:Picolines; D012685:Sepharose; D005982:Glutathione Transferase; D002918:Chymotrypsin; D005978:Glutathione; C033550:hexylglutathione; D003488:Cyanogen Bromide