Purification and characterization of class alpha and Mu glutathione S-transferases from porcine liver.
Six cytosolic GSTs from porcine liver were purified by a combination of glutathione affinity chromatography and ion-exchange HPLC. The isoenzymes were characterized by SDS-PAGE, gel filtration, isoelectric focusing, immunoblotting analysis and determination of substrate specificities and inhibition characteristics. The purified GSTs belong to the alpha and mu classes, respectively. No class pi isoenzyme was isolated or detected. The class alpha GST pA1-1* exists as a homodimer (M(r) = 25.3 kDa), whereas GST pA2-3* consists of two subunits with different M(r) values (27.0 and 25.3 kDa). The estimated pI values were 9.5 and 8.8, respectively. Furthermore, four class mu porcine GSTs, pM1-1*, pM1-2*, pM3-?* and pM4-?*, were isolated. The isoenzyme pM1-1* possesses a relative molecular mass of 27.2 kDa and a pI value of 6.2. Additional pM1 isoenzymes hybridize with the subunit pM2* (M(r) = 25.2) to furnish a heterodimer, which shows a pI value of 5.8. The other class mu isoenzymes are heterodimers with pI values of 5.45 and 5.05. Substrate specificities and inhibition characteristics correlate very well with those of the corresponding human isoenzymes. The results are discussed with regard to the usefulness of porcine GSTs as an in vitro testing model.
Kunze
T
T
Pharmazeutisches Institut, Christian Albrechts Universität, Kiel, Germany. tkunze@pharmazie.uni-kiel.de
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology*; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000145:classification / Q000276:immunology / Q000378:metabolism*; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D013552:Swine
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D005982:Glutathione Transferase