Approaches to In Vivo Inhibition of Glutathione S-Transferases

Abstract

1. Purification of rat liver Glutathione S-transferase. Affinity chromatography on bromosulphophthalein-glutathione conjugate convalently linked to Sepharose 4-B works well as a technique for preparing a purified glutathione S-transferase preparation, prior to separating the isoenzymes on a chromatofocusing column, with two reservations: 1)relatively low recovery of glutathione S-transferase off the affinity matrix, as a result of the salt wash required to produce an essentially pure glutathione S-transferase preparation; 2)GST2-2 binds relatively weakly to the matrix, resulting in loss of a major proportion of subunit 2 in the salt wash. 2. S-nitrosoglutathione. S-nitrosoglutathione is a moderately efficient dead-end inhibitor of GST1-1 and GST1-2 (Ki's of .0165 and .013mM respectively when glutathione was the varied substrate, .13 and .127mM respectively when CDNB was the varied substrate). The reversible nature of the inhibition suggests that catalysis by the glutathione S-transferase does not involve a covalently bound enzyme intermediate. It further indicates that the glutathione binds first in the enyzme catalysed reaction, suggesting either an ordered mechanism with glutathione binding first, or a random mechanism. 3. Triphenylmethane dyes. The triphenylmethane dyes can act as powerful inhibitors of the glutathione S-transferase, with the Ki ranging from .021mM for the inhibition of GST1-1 by Methyl Green, to an inhibition constant of 9.9x10-5mM for the inhibition of GST1-2 by Ethyl Violet. The partial non-competitive inhibition of the enzyme by all the triphenylmethane dyes provided evidence of a secondary binding site on the enzyme (the S-site), and suggested that interaction between inhibitors such as the triphenylmethane dyes and the S-site results in conformational changes in the enzyme. The inability of the triphenylmethane dye-glutathione adduct to bind effectively at the active site suggests that the H-site is inca pable of binding molecules which are not essentially planar. The H-site is likely to be in the form of a narrow hydrophobic slot rather than a more loosely defined hydrophobic cavity. Initial studies on the in vivo distribution of Malachite Green in rats after intra venous injection suggest that a sufficient quantity of the dye reach the liver to significantly inhibit the glutathione S-transferase, and partially deplete intracellular glutathione. 4. Trinitrobenzene. The Meisenheimer complex formed in aqueous solution between glutathione and 1,3,5-trinitrobenzene is the 2:1 complex, with an equilibrium constant of 1353±58 1.mole-1, and a molar extinction constant of 13270±244. 1,3,5-Trinitrobenzene was a poor non-competitive inhibitor when glutathione was the varied substrate and CDNB was the electrophilic substrate. No inhibition by Meisenheimer complex was noted between pH6.5 and pH9.0. When DCNB was the electrophilic substrate, inhibition by the Meisenheimer complex appeared to take place at pH9.0.