Abstract:
A methodology has been developed for the purification to homoqeneity of glutathione S-aryltransferase from Costelytra zealandica. However the Same purification sequence was shown to be ineffective for the enzyme from sheep liver. The techniques used were, successively, ammonium sulphate fractionation, affinity chromatography, DEAE cellu1ose (pH 7.3) chromatography, CM Cellulose (pH 7.3) chromatography, hydroxyapatite (HTP) chromatography and pH 4-10 isoelectric focusing (grass grub only). Affinity chromatography, using a substituted sepharose (GS-BSP-Sepharose) having as ligand a conjugate between GSH and the S-aryltransferase inhibitor bromosulphonphthalein, played the key role in the purification sequence. After HTP chromatography the insect and vertebrate enzymes had been purified 149 and 5 fold respectively over the ammonium sulphate fractions. At this stage of purification, the grass grub enzyme was shown to run as two active protein bands on both pH 8.3 disc gel electrophoresis and pH 4-10 gel isoelectric focusing. The same sample ran as a single protein band corresponding to a molecular weight of 27,000 on sodium dodecyl sulphate gel electrophoresis. In contrast, pH 4-10 gel isoelectric focusing of the Sheep liver enzyme showed the presence of at least 14 protein staining bands. Subsequent pH 4-10 preparative column isoetectric focusing of the grass grub enzyme from HTP resolved the Sample into two components; a minor one of pI 5.9 and a major one of pI 8.7. The latter appeared homogeneous by criteria of both disc gel electrophoresis and pH 4-10 gel isoelectric focusing.
The pI 5.9 and pI 8.7 fractions were shown to have overlapping substrate specificities. The major differences between them were the absence of, detectable aralkyl.- (p-nitrobenzyl chloride) activity in the pI 5.9 fraction and of aryl-( 1,2-dichloro-4-nitrobenzene) ) and alkene- (S-crotonyl-N-acetylthioethanolamine) activities in the pI 8.7 fraction.
The temperature stability of the insect and vertebrate enzymes in the presence and absence of, 1 mM EDTA, lmM glutathione, and 30% glycerol Was examined. The additives were shown to confer stability to the enzyme from both sources only at elevated temperature.
Ion exchange chromatography (DEAE- and CM- cellulose) of, the sheep liver enzyme was studied, over the pH range 5-9.
The binding of the grass grub enzyme to the GS-BSP-Sepharose support was investigated in detail. Apparent differences were noted between the binding and non-binding fractions and these fractions further studied by pH 4-10 isoelectric focusing. A tentative suggestion is made that, of the three major grass grub activities (of pI 4.6, 5.9 and 8.7), only the pI 5.9 and 8.7 forms are bound by the affinity support. Some evidence is presented for the interconversion between these enzyme forms
