Further analysis of multiple forms of rabbit hepatic glutathione S-transferase.

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Abstract
In the previous study, carboxymethyl (CM)-cellulose chromatography of a rabbit liver extract gave at least four glutathione S-transferase activity peaks (peaks R1, R2, R3 and R4). Further purification of peaks R2 and R3 by S-hexylglutathione Sepharose 6B and hydroxylapatite chromatography resolved peak R2 into one peak (a homodimer of the Y2Y2 subunits; Y2 Mr 25000), and peak R3 into two peaks, R3a (a heterodimer of the Y1Y3 subunits; Y1 Mr 24500; Y3 Mr 26500) and R3b (a homodimer of the Y3Y3 subunits). In the present study, isoelectric focusing was used to separate the diethylaminoethyl (DEAE)-flow-through fraction into at least eight activity peaks (pIs 10.95-10.77, 10.35, 9.95, 9.40, 8.88, 8.48, 8.36 and 7.66), which were designated as activity peaks I, II, III, IV, V, VI, VII and VIII in decreasing order of isoelectric point. On the other hand, these peaks could be resolved into at least seven activity peaks by convenional CM-cellulose chromatography, the four main activity peaks of which were assigned as peaks R1, R2, R3 and R4 reported previously. As a result of the individual isoelectric focusing of peaks R1-R4, activity peak I having the highest and broadest isoelectric point was identified as peak R4. By sodium dodecyl sulfate (SDS)/polyacrylamide-gel electrophoresis, peaks II, III and VII were identified as the previously characterized R3a, R3b and R2, respectively. The isoelectric focusing of peak R1 gave four activity peaks (pIs 9.72, 8.56, 7.64 and 7.07). The enzyme with pI 9.72 was found to be a homodimer of the Y1Y1 subunits and the other three enzymes were all heterodimers of Y2 subunit and a new type of subunit (designated as Y4: Mr 28000). Among these four peaks, the enzymes with pIs 9.72 and 7.07 could not be observed in the isoelectric focusing pattern of the gross DEAE-flow-through fraction. The enzymes with pIs 8.56 and 7.64 were identified as peaks VI and VII, respectively, on the basis of SDS/polyacrylamide-gel electrophoresis and their isoelectric points.