Purification and Characterization of the Glutathione-S-transferases from the Northern Quahog Mercenaria mercenaria

Glutathione-S-transferase (GST) was isolated from the northern hardshell clam Mercenaria mercenaria (quahog) using a two-step procedure involving ammonium sulfate precipitation and affinity chromatography. Kinetic analysis of the purified enzyme using 1-chloro-2,4-dinitrobenzene as substrate revealed a specific activity of 38.0 mu mol/min/mg, while V sub(max) and K sub(m) values were estimated as 48.0 mu mol/min/mg and 0.24 mM, respectively. Electrophoretic analysis of GST indicated multiple forms of the dimeric enzyme in quahogs with subunit molecular masses of 22, 24, 25, and 27 kDa. Isoelectric focusing analysis resulted in pI values for three isoenzymes of 5.1, 4.9, and 4.6. The acidic pI values obtained indicated that quahog GST belongs to the pi class. Inhibition of quahog GST by tetrapyrroles was similar to that of GST from oyster and rat liver. Quantitative comparison of tetrapyrrole inhibition patterns of quahog GST with those of oyster and rat liver GST indicated lower inhibition rates by three of the four tetrapyrroles tested (bilirubin, biliverdin, and chlorophillyin), suggesting that quahog GST could differ structurally or functionally from oyster and rat liver GSTs.