Characterization of rat pancreatic glutathione S-transferases by chromatofocusing, reverse-phase high-performance liquid chromatography, and immunohistochemistry.
The cytosolic glutathione S-transferases (GSTs) are a family of phase II detoxifying isoenzymes that catalyze the interaction of the tripeptide thiol glutathione (GSH) with a wide variety of reactive and often toxic or carcinogenic electrophilic substrates. Pancreatic GSTs, however, have only been partially characterized. In this study, pancreatic cytosolic GSTs from male Fisher 344 rats were semipurified by affinity chromatography and then analyzed for isoenzyme content by chromatofocusing (fast protein liquid chromatography) and for subunit content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. In addition, polyclonal rabbit antisera were produced against homodimeric isoenzymes purified from rat liver and kidney, including the alpha class isoenzymes 1-1 and 2-2, the mu class isoenzyme 4-4, and the pi class isoenzyme 7-7. These antisera were used in immunohistochemical (IHC) studies of the distribution of the pancreatic GSTs. A range of 0.5-1.6% of the total protein in rat pancreatic cytosol was found to be GST protein. The most abundant subunits present were the pi subunit 7 and mu subunits 3 and 4. Using modified methodology, smaller amounts of the alpha subunit 2 and the mu subunit 6 were detected, whereas very small amounts of the alpha subunits 1 and 8 were present. The IHC demonstrated that the GSTs were in large part limited to the duct system of the exocrine pancreas, with positive staining of endothelial cells and stroma observed for the alpha and mu subunits. Isoenzymes containing the alpha subunit 2 were preferentially expressed in centroacinar cells and small ductules, whereas those containing the mu subunit 4 and the pi subunit 7 were more prevalent within larger ductules and ducts. The lumens of the largest ducts also contained the two subunits 4 and 7. It is concluded that the acinar cells of the exocrine pancreas may lack the protection against electrophilic toxic and carcinogenic agents provided by the ductular system by GSTs.
March
T H
TH
Department of Veterinary Pathobiology, University of Illinois, Urbana 61802, USA.
Jeffery
E H
EH
Wallig
M A
MA
D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000032:analysis*; D007124:Immunoenzyme Techniques; D008297:Male; D010179:Pancreas / Q000166:cytology / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344
D005982:Glutathione Transferase