On the identity of the estrogen-sensitive enzyme of human placenta.

Our previous experiments on the mechanism of action of estradiol (l-5) had led to the hypothesis that the soluble protein fraction (Ssr,ooo) of homogenates of human plac.enta contains a DPNl-linked isocitric dehydrogenase that is activated by minute amounts of estrogen added in vitro. The same fraction when incubated with TPN instead of DPX showed marked TPN-linked isocitric dehydrogenase activity, but the rate of this reaction was unaffected by the addition of estradiol. In the course of attempts to purify the estrogen-sensitive enzyme, it was found that the protein precipitated from Srr,ooo by 30 per cent saturation with ammonium sulfate had no activity alone; it would not oxidize isocitrate when DPN was present in either the presence or the absence of estradiol. The protein precipitated by between 30 to 60 per cent saturated ammonium sulfate contained what appeared to be a DPN-linked isocitric dehydrogenase that showed only a slight response to added estrogen. However, when these two fractions were mixed together and isocitrate was added as the substrate, there was rapid DPS reduction in the presence of estradiol and much less DPN reduction when no estradiol was added. ATP was found to increase greatly the magnitude of the estrogen effect, and ADP was recovered from the incubation material at the end of the incubation period and identified by paper chromatography. Further attempts at purification were consistently unsuccessful. These data were interpreted as being consistent with the theory that the 0 to 30 per cent ammonium sulfate fraction contained an “activating enzyme,” that the 30 to 60 per cent fraction contained the isocitric dehydrogenase, and that the hormone stimulated the activating enzyme to convert an inactive form of the isocitric dehydrogenase to an active form. This was analogous to the theory postulated by Rall et al. (6) on the activation of phosphorylase by epinephrine. Talalay and Williams-Ashman (7) confirmed our observation that estrogen stimulates the isocitric dehydrogenase system. They found that catalytic amounts of TP1;T are required for restoring the activity of placental preparations that do not respond to estrogens. They further found that an estrogen stimulation of the dehydrogenation of glucose-6-phosphate was