Purification and characterization of multiple forms of rabbit hepatic glutathione S-transferase.

Publisher Website
Google Scholar
Abstract
Multiple forms and properties of glutathione S-transferase were investigated by using the liver from individual male or female rabbits. Most of the activity toward 1-chloro-2,4-dinitrobenzene (CDNB) in the crude extract passed through a diethylaminoethyl (DEAE)-cellulose column. On carboxymethyl (CM)-cellulose chromatography, the flow-through fraction was resolved into at least four activity peaks which were designated as R1, R2, R3 and R4 in order of elution. The content of each component varied among the individual rabbit livers examined. The main components, R2 and R3, were further purified to homogeneity as judged by sodium dodecyl sulfate (SDS)/polyacrylamide-gel electrophoresis. R3 contained two forms, which were named R3a and R3b in order of elution from a hydroxylapatite column. R2, R3a and R3b each had a molecular weight of approximately 51000 as estimated by gel filtration. R2 and R3b appeared to be homodimers consisting of subunits of apparently different size (R2, Y2: 25000; R3b, Y3: 26500). On the other hand, R3a was a heterodimer composed of subunits with molecular weights of 24500 (Y1) and 26500 (Y3). The molecular weight of the subunit of R3b was identical with that of Y3. These enzymes showed the highest activity toward CDNB and much lower activities toward the other substrates tested, and the substrate specificities of all the enzymes were roughly similar to each other. Our highly purified enzyme forms were compared with rat hepatic glutathione S-transferases with regard to substrate specificity, subunit species, kinetic parameters, isoelectric point and amino acid composition.