The reaction of aralkyl sulphate esters with glutathione catalysed by rat liver preparations

Abstract

1. Rat liver supernatant preparations catalyse the reactions of some aralkyl sulphate esters with GSH to yield S-aralkylglutathione derivatives. 2. A glutathione S-transferase that catalyses these reactions has been purified 16-fold. 3. The purified enzyme preparation catalyses the release of sulphate ions from benzyl sulphate, 1-menaphthyl (naphth-1-ylmethyl) sulphate and phenanthr-9-ylmethyl sulphate only in the presence of GSH. It does not cause the release of sulphate ions from prop-1-yl sulphate, l-serine O-sulphate, phenyl sulphate or oestrone 3-sulphate even when GSH is added. 4. The stability and specificity of the enzyme and its response to inhibitors and to changes of pH were studied. 5. The activity of the preparation was compared with the activities of glutathione S-transferases described previously.

The mechanism of the reaction between glutathione and 1-menaphthyl sulphate catalysed by a glutathione S-transferase from rat liver

“…The biuret method as modified by Cleland & Slater (1953) was used, as described by Gillham (1971), for the determination of protein in the first five stages of the enzyme purification. The protein present in the final two stages of the purification was measured bytheextinction at 280nm: E"' 1.0/2.9mg of protein in 3ml (calibrated by comparison with biuret samples).…”

“…The assay method was detailed by Gillham (1971). All measurements were made in 50mM-triethanolammonium chlorideNaOH buffer, pH8.3, at 37°C.…”

“…The method of purification of the rat liver enzyme that catalyses the reaction of 1-menaphthyl sulphate with GSH has been modified and extended from the method given by Gillham (1971). The enzyme was separated from the livers of 24 rats instead of eight.…”

“…As a result of this scaling-up the stage (1) extract was obtained by centrifuging the rat liver homogenate for 1 h at 45000g. Stages (2)-(4) were carried out as described by Gillham (1971 In stage (7) the active fractions from stage (6) were concentrated by positive-pressure dialysis to 5ml. The concentrate was applied to a column (2.5cm x 60cm) of Sephadex G-200, which had been equilibrated with SOmM-KH2PO4-NaOH buffer, pH7.6.…”

“…Indeed few attempts have been made to treat glutathione S-transferases kinetically as twosubstrate enzymes. A glutathione S-transferase was purified from rat liver (Gillham, 1971) and was shown to catalyse the reaction of GSH with acid-sulphate esters of aralkyl alcohols to form SO42-and the corresponding S-aralkylglutathione. In a preliminary study ofthe enzyme based on the assumption that the reaction sequence is random, i.e.…”