Abstract:
Glutathione S-transferase isoenzymes from Musca domestica pupae were purified and separated through the use of affinity chromatography followed by isoelectrofocusing. Two groups, having distinct substrate specificities, were distinguished through affinity chromatography. The BSP-bound group, exhibiting activity toward DCNB and EPNP, could not be purified further. The glutathione-bound group was shown to have a high activity with CDNB, and detectable activity toward ethacrynic acid and EPNP.
The glutathione-bound isoforms were separated through isoelectrofocusing into four isoenzymes with subunit molecular mass between 23.8 and 27.3kD. Their respective isoelectric points were determined as 4.65, 5.53, 6.18 and 6.72. Kinetic analysis indicated a rapid equilibrium random Bi Bi mechanism for these isoenzymes, but no functional differences between them were able to be shown.
Examination of the reaction progress curves of the four low pI isoenzymes, using the method of Yun and Suelter, indicated that they are subject to a complex form of product inhibition. A mechanism for this inhibition is proposed.
From this work it would appear that the expression of glutathione S-transferase enzymes in pupal Musca domestica is different from that at other lifecycle stages.
