RAT-LIVER THETA-CLASS GLUTATHIONE S-TRANSFERASES T1-1 AND T2-2 - THEIR CHROMATOGRAPHIC, ELECTROPHORETIC, IMMUNOCHEMICAL, AND FUNCTIONAL-PROPERTIES

A method was established for simultaneously isolating Theta-class glut athione (GSH) S-transferases (GSTs) T1-1 and T2-2 as homogeneous prote ins from rat (r) liver cytosol. The established method of using an 8-a minooctyl Sepharose 4B column to separate rGSTT1-1 from rGSTT2-2 at th e final stage of their purification was a modification of the method p reviously reported for the isolation of rGSTT2-2 (Hiratsuka et al., J. Biol. Chem., 265, 11973-11981, 1990). Specific substrates used for pu rification of the Theta-class rGSTs were dichloromethane for T1-1 and 5-sulfoxymethylchrysene for T2-2. rGSTsT1-1 and T2-2 existed at a rati o of 1:7 at a total concentration of 0.5% of that of the cytosolic pro tein, Purified rGSTsT1-1 and T2-2 were separated as single bands at 28 and 26.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophor esis and as single peaks at retention times of 36 and 34 min, respecti vely, by reverse-phase partition high-performance liquid chromatograph y on a mu Bondasphere column eluted with a linear gradient of acetonit rile in water containing trifluoroacetic acid. Western blot analysis i ndicated that rabbit antisera raised against rGSTsT1-1 and T2-2 intens ely reacted with the corresponding antigens, but showed no detectable reactivity with the different isoforms of Theta-class rGSTs as well as with representative hepatic rGSTs of other classes. The Theta-class r GSTs showed higher GSH peroxidase activity than rGSTA1-2 toward hydrop eroxides of cumene, arachidonic acid, and linoleic acid. Cumene hydrop eroxide was a better substrate for rGST T1-1 than for rGST T2-2, while the fatty acid hydroperoxides were the better substrates for rGST T2- 2 than for rGST T1-1. (C) 1997 Academic Press.