Purification and characterization of a hepatic mitochondrial glutathione S-transferase exhibiting immunochemical relationship to the alpha-class of cytosolic isoenzymes.
Hepatic mitochondria from different mammalian species contain varying levels of glutathione S-transferase (GST) activities. More than 70% of the activity detectable in the mouse liver mitochondria is associated with the soluble matrix. The mouse mitochondrial matrix GST was purified using a combination of (NH4)2SO4 fractionation, Sephadex gel filtration and affinity chromatography on glutathione (GSH) conjugated Sepharose. The purified GST comigrates with the mouse cytosolic MI (or alpha form), and exhibits an apparent molecular mass of 25 kD on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antibody to the purified mitochondrial GST cross-reacted with the similarly migrating cytosolic MI GST, suggesting extensive immunochemical relatedness between these two forms. As previously demonstrated for the cytosolic alpha form, the mitochondrial GST catalyzes aflatoxin B1-GSH conjugation (6.3 nmol/mg protein/min) and exhibits peroxidase activity (6.7 mumol/mg protein/min). The putative mitochondrial GST only in intact mitochondria, but not in sonic disrupted mitochondria, is resistant to proteolytic digestion with trypsin, demonstrating its intramitochondrial location. Isoelectric focusing on the flat bed polyacrylamide gel system resolves the mitochondrial GST into two distinct components with apparent pI of 9.9 and 9.7, both of which cross-react with polyclonal antibody to the mitochondrial GST. Under the identical conditions, the most cationic form of cytosolic GST cross-reacting intensely with the antibody resolves as a single component with an apparent pI of 9.4. Thus the mitochondrial GST resembles the alpha family of isoenzymes, though it appears to represent independent molecular species different from the cytosolic forms.
Addya
S
S
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6047.
Mullick
J
J
Fang
J K
JK
Avadhani
N G
NG
D016604:Aflatoxin B1 / Q000378:metabolism; D000818:Animals; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000145:classification / Q000276:immunology / Q000302:isolation & purification*; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D013379:Substrate Specificity; D015262:Xenobiotics / Q000378:metabolism
D007527:Isoenzymes; D015262:Xenobiotics; D016604:Aflatoxin B1; D005982:Glutathione Transferase; D005978:Glutathione