H.p.l.c. separation and study of the charge isomers of @entity1 placental XXXX transferase.

@entity1 @entity2041 @entity2197 @entity2975 @entity7734
@entity2041 transferase (GST) from @entity1 placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of @entity2197 , suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the @entity2197 concentration, @entity2975 -gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for @entity2197 was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using @entity7734 were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of @entity2197 . Two apparent Km values of 0.38 and 50.27 mM were obtained for @entity2197 , whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low @entity2197 concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high @entity2197 concentration. Since the average tissue concentration of total @entity2197 was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo.
