Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat

Abstract

The glutathione S-transferases are dimeric enzymes whose subunits can be defined by their mobility during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as Yf (Mr 24,500), Yk (Mr 25,000), Ya (Mr 25,500), Yn (Mr 26,500), Yb1 (Mr 27,000), Yb2 (Mr 27,000) and Yc (Mr 28,500) [Hayes (1986) Biochem. J. 233, 789-798]. Antisera were raised against each of these subunits and their specificities assessed by immuno-blotting. The transferases in extrahepatic tissues were purified by using, sequentially, S-hexylglutathione and glutathione affinity chromatography. Immune-blotting was employed to identify individual transferase polypeptides in the enzyme pools from various organs. The immuno-blots showed marked tissue-specific expression of transferase subunits. In contrast with other subunits, the Yk subunit showed poor affinity for S-hexylglutathione-Sepharose 6B in all tissues examined, and subsequent use of glutathione and glutathione affinity chromatography. Immuno-blotting was employed to identify a new cytosolic polypeptide, or polypeptides, immunochemically related to the Yk subunit but with an electrophoretic mobility similar to that of the Yc subunit; high concentrations of the new polypeptide(s) are present in colon, an organ that lacks Yc.

Glutathione S-transferase expression in the human testis and testicular germ cell neoplasia

“…Hayes, University Department of Clinical Biochemistry, Edinburgh. Isoenzyme preparation, rabbit immunisation and characterisation of antibodies have been previously described (Morgenstern et al, 1990;Hayes et al, 1983;Hayes et al, 1987;Hayes et al, 1986).…”

Distribution of glutathione S-transferase isoenzymes in human ovary

“…Multiple forms of GSTs originate from dimeric combination of different subunits (Habig et ai, 1974;Hayes & Mantle, 1986). There are three distinct homodimeric or heterodimeric classes of cytosolic GST ( , and µ) in man (Hayes et ai, 1987) and one trimeric, membrane-bound form known as microsomal GST (Morgenstern & De Pierre, 1986).…”

“…Polyclonal antisera to GST isoenzymes were used. The preparation of GST antisera from purified human isoenzymes, rabbit immunization schedule and assessment have been described previously (Hayes & Mantle, 1986;Hayes et ai, 1987). These antisera reacted specifically with GSTn, and µ and microsomal GST.…”

Glutathione S-transferase expression in fetal kidney and Wilms' tumour

“…It comprises undifferentiated blastema containing differentiated tubules and glomeruloid forms (Lawler et al, 1977;Beckwith & Palmer, 1978) which morphologically and biochemically show similarities to the embryonic metanephros which forms the kidney (Mierau et al, 1987;Roth et al, 1988). Nephroblastoma generally responds well to chemotherapy (Lawler et al, 1977 (Hayes et al, 1983(Hayes et al, , 1987Hayes & Mantle, 1986;McLellan et al, 1989). The antibodies did not show any cross reactivity.…”

“…Three distinct cytosolic isoenzyme groups exist, referred to as classes alpha, mu and pi GST, or GST I, II and III (Mannervik, 1985;Hayes & Mantle, 1986). Recently a distinct human microsomal GST has been described and purified (McLellan et al, 1989).…”