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 protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and 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. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.
Bohets
H H
HH
I.H.E., Afdeling Toxicologie, Brussel, Belgium.
Nouwen
E J
EJ
De Broe
M E
ME
Dierickx
P J
PJ
D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology*; D004285:Dogs; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007526:Isoelectric Point; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007672:Kidney Cortex / Q000166:cytology / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D018675:Toxicity Tests
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D005982:Glutathione Transferase