Partial purification and characterization of methyl-p-hydroxyphenyllactate esterase in rat uterine cytosol

Abstract

Previous studies from this laboratory have shown that methyl-p-hydroxyphenyllactate (MeHPLA) is a bioflavonoid and/or tyrosine metabolite that inhibits both normal and malignant cell proliferation, presumably, by association with nuclear type II [ 3H]estradiol binding sites. Conversely, the corresponding free acid, phydroxyphenyllactate (HPLA) possesses little, if any, cell regulatory activity. Therefore, factors that control the relative concentrations of MeHPLA and HPLA in normal or malignant mammalian tissues may also influence the rate of cellular proliferation. The experiments in this manuscript describe the characterization and purification of MeHPLA esterase from the rat uterus. These studies demonstrate that MeHPLA esterase activity is relatively homogeneous, eluting as a single peak during DEAE ion exchange chromatography and phenyl agarose hydrophobic interaction chromatography. Affi-gel Blue (Cibacron Blue F3GA) affinity chromatography resulted in a significant purification (approx. 300-fold) of the MeHPLA esterase activity, and a combination of these chromatographic steps resulted in a significant purification (>4300-fold) of this protein. Further analysis of these esterase preparations on nondenaturing agarose gels allowed us to visualize bands of esterase activity. These studies localized one major low-mobility band of esterase activity in rat uterine cytosol preparations, which was increased by estradiol treatment. Furthermore, estrogen induction of MeHPLA esterase in the rat uterus was completely blocked ( p < .01) by luteolin, although this bioflavonoid did not affect esterase activity in cytosol preparations from nonestrogenized rats. These results confirm our earlier studies demonstrating that MeHPLA hydrolysis is under estrogen regulation and that bioflavonoids antagonize estrogen action through pathways involving type II site induction and/or the regulation of MeHPLA hydrolysis in normal and malignant cells.