Characterization of cytosolic estrogen sulfotransferase from RL95-2 endometrial cancer cells.

Estrogen sulfoconjugation previously was reported in normal endometrium and in RL95-2 cells, a cell line derived from a human endometrial cancer maintained in continuous in vitro culture. In the present study the estrogen sulfurylation activity in the cytosolic fraction of RL95-2 cells was characterized using [3H]estrone as substrate. Estrone sulfate was separated from unreacted estrone by thin layer chromatography. Activity was proportional to cytosol concentration, with a pH optimum at pH 8. There was marked temperature dependence between 24 and 40 C. The apparent Km for estrone conjugation was 3.6 nM, with a maximum velocity of 135 fmol/micrograms DNA . h. No complex kinetic behavior was found at estrone concentrations up to 1 microM. The apparent Km for the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate was 0.6 microM. Inhibition experiments demonstrated that the sulfurylating activity studied under these conditions was specific for estrogens. Only estradiol and estriol, in addition to estrone itself, inhibited conjugation to any significant degree. Dehydroepiandrosterone had only 1% the inhibitory activity of estrone. Other androgens, corticoids, progestins, phenols, nonsteroidal estrogens and antiestrogens, and bile acids had no significant effects on the sulfurylation of estrone. An estrogen-sulfoconjugating activity with the characteristics of estrogen sulfotransferase (EST) was demonstrated in RL95-2 cells. The Km of EST for estrone in the RL95-2 cells closely approximated the value reported for the enzyme in normal endometrium. The affinity of EST for estrogens is within the range of the Kd of estrogen receptor and of the physiological concentrations of estrogens reported in the endometrium, suggesting that EST could serve as a regulator of intracellular estrogen levels.