Purification and biochemical characterization of the rabbit pulmonary glutathione S-transferase: stereoselectivity and activity toward pyrene 4,5-oxide.
Two homodimeric isozymes, glutathione S-transferase (GST) 25 kDa and GST 27 kDa, in equal proportion comprise the majority (greater than 75%) of the pulmonary cytosolic GST of untreated rabbits. The subunits of GST 25 kDa and GST 27 kDa are distinguishable by electrophoretic mobility (25 and 27 kDa, respectively), apparent isoelectric points (pI 7.4 and pI 9.1, respectively), and immunoreactivity. Immunoblots indicated that these subunits may be minor components in hepatic cytosol. The pulmonary isozymes could not be distinguished by their activities toward chloro-2,4-dinitrobenzene (CDNB) or activity and stereoselectivity toward pyrene 4,5-oxide (PyO). The purified GST fractions represented less than or equal to 16% of the PyO activity for pulmonary cytosol. The stereoselectivity of the cytosolic GST for the pro-S-configured oxirane carbon of PyO was not maintained in the purified preparations which were virtually nonstereoselective. Immunoprecipitation of pulmonary cytosolic GST with anti-GST 27 kDa and anti-GST 25 kDa indicated that at least 84 and 60% of the activity toward CDNB and PyO, respectively, is mediated by the two isozymes. The specific PyO activities of GST 27 kDa, GST 25 kDa, and the rabbit hepatic preparations (approximately 0.2 unit/mg) were similar to that of hepatic GST purified from horse, cow, and pig, and to human placental GST pi (0.02-0.5 unit/mg) but one-tenth that of rat hepatic GST or human hepatic GST mu. However, the activity of the hepatic cytosol from rat and human was similar to that of rabbit. Thus, some GST isozymes may be particularly susceptible to modulation of activity/stereoselectivity that can be discerned with arene oxide substrates such as PyO.
Serabjit-Singh
C J
CJ
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Bend
J R
JR
D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000302:isolation & purification*; D006736:Horses; D006801:Humans; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology; D011721:Pyrenes / Q000378:metabolism*; D011817:Rabbits; D013552:Swine
D011721:Pyrenes; C049292:pyrene 4,5-oxide; D005982:Glutathione Transferase