THE CYTOSOLIC GLUTATHIONE-S-TRANSFERASE ISOENZYMES IN THE DOG KIDNEY CORTEX AS COMPARED WITH THE CORRESPONDING MDCK RENAL-CELL LINE

Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protei n, for dog and MDCK, respectively. Cytosolic GST was only partially pu rified by glutathione affinity chromatography, a substantial amount (4 3% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separat ed into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDC K, respectively. Flow-through GST was purified by gel filtration, anio n exchange chromatography and anionic chromatofocusing showing only on e GST isoenzyme, with distinct features from the affinity bound GST, f or both dog and MDCK. The isoenzymes were characterized by their kinet ic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to t he MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a compara ble affinity towards GSH and comparable sensitivities towards the inhi bitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue an d hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibit or and substrate sensitivities showed that the affinity bound GSTs bel ong to class pi and mu, the presence of class pi was confirmed by immu noblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a near ly neutral pI, a high affinity towards CDNB and an equal sensitivity t owards triphenyltin chloride, cibacron blue and hematin. However, base d on inhibitor studies and immunoblot, this isoenzyme could not be att ributed to an identified GST class. The overall isoenzyme pattern of d og and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all bel ong to class mu or pi.