Characterization of Glutathione S-Transferases from the Housefly Musca Domestica (L)

Abstract

1) A new support for the affinity chromatography of glutathione S-transferases, glutathione covalently linked to epichlorohydrin-activated Sepharose, adsorbs specifically a group of glutathione S-transferases from the housefly. Those isoforms not adsorbed to this matrix do bind to the glutathione conjugate of the dye sulfobromophthalein, covalently bound to Sepharose. An effective method for the isolation of housefly glutathione S-transferases has been devised which involves the sequential use of these two affinity supports. 2) The iso-enzymes binding to immobilized glutathione may be further separated by isoelectric focusing (but are inactivated by chromatofocusing) and are acidic, having isoelectric points less than pH 6.5. These isoenzymes are characterized by having catalytic activity with ethacrynic acid and having little or no activity with 1,2-dichloro-4-nitrobenzene. The isoenzymes binding to the immobilized glutathione - sulfobromophthalein conjugate may be separated by chromatofocusing. They are basic, having isoelectric points of greater than 7.5. They are particularly characterized by catalytic activity with 1,2-dichloro-4-nitrobenzene, by a high activity with trans-4-phenyl-3-butene-2-one and by negligible activity with ethacrynic acid. Both groups of enzymes have comparable activities with 1-chloro-2, 4-dinitrobenzene and p-nitrobenzyl chloride. 3) The purified and partially purified isoenzymes in both classes were found to be unstable. In the absence of stabilizing agents, both groups of isoenzyme lost activity on freezing, freeze drying or vacuum dialysis. Enzyme preparations kept at 4°C lost activity more slowly than those frozen and thawed. Inclusion of dithiothreitol (1mM) and mannitol (10 mM) was found to increase the stability of the enzyme preparations. Inclusion of glycerol (up to 30% v/v) in enzyme solutions protected against the effects of freezing. 4) Isoenzymes in the acidic group were found to be dimeric and to consist of subunits from one or two groups of molecular weights of 23.7 kD and 27.4 kD. Amino acid analysis of the N-terminal of polypeptides from these two groups revealed those from the 23.7 kD group had a blocked N-terminus. The N-terminal sequence from the higher M group was heterogeneous at two sites in the first fourteen amino acids, suggesting the presence of up to nine closely related polypeptides. The sequence showed no homology with that of known glutathione S-transferases from other sources. The high pI groups of isoenzymes were dimers containing subunits from one or two groups of polypeptides of molecular weights of 27.3 kD and 25.1 kD. These subunits were not characterized further. 5) The two groups of isoenzymes were further characterized with respect to their response to two classes of inhibitor. Organic compounds, which have been used to characterize mammalian glutathione S-transferases were tested. The compounds were bromosulfophthalein, haematin, Cibacron Blue, and triphenyl tin chloride. Values for I50 for these inhibitors were consistently greater for the high pI group of isoenzymes than for the low pI group. Six inorganic anions (chloride, bromide, iodide, nitrate, thiocyanate and perchlorate) were tested as alternate product inhibitors. The order of inhibitory power was thiocyanate ≥ perchlorate > iodide ≥ nitrate > bromide > chloride. The isoenzyme of highest pI was least susceptible to inhibition. There was not a clear trend amongst the other isoenzymes. 6 ) The kinetics of the various isoenzymes with respect to substrate concentration was examined. For the low pI set of enzymes, VM and KCDNB values were generally consistent (approx. 60 μmol/min/mg and 0.5 mM respectively) whereas KGSH varied over a wide range (0.06 - 1.2 mM). For the high pI isoforms, VM values varied over a five-fold range (8-40 μmol/min/mg) and values for KGSH and KCDNB varied over a three-fold range (1.5 - 5.1 and 0.45 - 1.5 mM respectively). The isoform of intermediate pI had the highest Michaelis oonstants with respect to both substrates. 7. Housefly larvae were fed with butylated hydroxy toluene in their diet. This treatment caused induction of both glutathione S-transferase activity with respect to both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. Activity with respect to the former substrate rose by a factor of 1.5x, and by 3.6x with respect to the latter.