Isolation and characterization of glutathione-S-transferase isozyme Q3 from the northern quahog, Mercinaria mercinaria.
Three glutathione-S-transferase (GST) isozymes (Q1, Q2, and Q3) from the northern quahog (Mercinaria mercinaria) were purified and separated with a combination of affinity and ion exchange chromatography. SDS-PAGE analysis of the separated quahog GSTs indicated there are four distinct subunits of the enzyme with molecular masses ranging between 23 and 27 kDa. The electrophoretic analysis in combination with GST information from literature indicates that among the quahog GST isozymes, there is a single homodimer and two heterodimers. Enzymatic kinetic analysis of the homodimeric quahog GST (Q3) using 1-chloro-2,4-dinitrobenzene and glutathione as reactants resulted in Vmax and Km values of 33.2 micromol min(-1) mg(-1) and 0.40 mM, respectively. A pH profile analysis of the Q3 GST indicates that the optimum catalytic pH is 7.6. The Q3 isozyme composes about 28% of the ion exchange purified GSTs but accounts for only 9% of the total GST enzymatic activity (25 micromol min(-1) mg(-1). An analysis investigating the dependence of the Q3 GST activity on temperature resulted in a retention of enzymatic activity (50-30% at temperature extremes from -13 degrees C to 100 degrees C), suggesting a unconventional role for the Q3 GST in quahog metabolism.
Blanchette
Brian N
BN
Department of Chemistry and Biochemistry, University of Massachusetts, Dartmouth 02747, USA.
Singh
Bal Ram
BR
D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008658:Inactivation, Metabolic; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D021122:Protein Subunits; D013696:Temperature
D007527:Isoenzymes; D021122:Protein Subunits; D005982:Glutathione Transferase