STEROID SEX HORMONES 



657 



estrogen-sensitive enzyme is a transhydro- 

 genase, as the evidence now indicates. The 

 results of kinetic studies with the coupled 

 isocitric dehydrogenase-transhydrogenase 

 system are consistent with this theory 

 (Gordon and Villee, 1955; Villee, 1957b; 

 Hagerman and Villee, 1957). Apparent 

 binding constants for the enzyme-hormone 

 complex (Gordon and Villee, 1955j and for 

 enzyme-inhibitor complexes have been cal- 

 culated (Hagerman and Villee, 1957). 



The observation that estradiol and es- 

 trone, which differ in structure only by a 

 pair of hydrogen atoms, are equally ef- 

 fective in stimulating the reaction suggested 

 that the steroid might be acting in some way 

 as a hydrogen carrier from substrate to 

 pyridine nucleotide (Gordon and Villee, 

 1956). Talalay and Williams-Ashman 

 (1958) suggested that the estrogens act as 

 coenzymes in the transhydrogenation reac- 

 tion and postulated that the reactions were: 



Estrone + TPNH + H* 



— Estradiol + TPN^ 



Estradiol + DPN+ 



— Estrone + DPNH + H* 



Sum : TPNH 



H* 



- DPN^ 

 — TPN^ + H^ 



DPNH 



This formulation implies that the estro- 

 gen-sensitive transhydrogenation reaction 

 is catalyzed by the estradiol-17y3 dehydro- 

 genase characterized by Langer and Engel 

 (1956). This enzyme was shown by Langer 

 (1957) to use either DPN or TPN as hy- 

 drogen acceptor but it reacts more rapidly 

 with DPN. Ryan and Engel (1953) showed 

 that this enzyme is present in rat liver, and 

 in human adrenal, ileum, and liver. How- 

 ever, no estrogen-stimulable enzyme is 

 demonstrable in rat or human liver (Villee, 

 1955). The nonparticulate fraction obtained 

 by high speed centrifugation of homogen- 

 ized rabbit liver rapidly converts estradiol 

 to estrone if DPN is present as hydrogen 

 acceptor, but does not contain any estrogen- 

 stimulable transhydrogenation system. 



It will not be possible to choose between 

 these two hypotheses until either the estro- 

 gen-sensitive transhydrogenase and the 

 estradiol dehydrogenase have been sepa- 

 rated or there is conclusive proof of their 



identity. Talalay, Williams-Ashman and 

 Hurlock (1958) reported a 100-fold puri- 

 fication of the dehydrogenase without sepa- 

 ration of the transhydrogenase activity 

 and found that both activities were in- 

 hibited identically by sulfhydryl inhibitors. 

 In contrast, Hagerman and Villee (1958) 

 obtained partial separation of the two ac- 

 tivities by the usual techniques of protein 

 fractionation, and reported that a 50 per 

 cent inhibition of transhydrogenase is ob- 

 tained with p-chloromercurisulfonic acid at 

 a concentration of 10~^ m whereas 10"^ m 

 p-chloromercurisulfonic acid is required for 

 a 50 per cent inhibition of the dehydro- 

 genase. The evidence that these two ac- 

 tivities are mediated by separate and dis- 

 tinct proteins has been summarized by 

 Villee, Hagerman and Joel (1960). 



The transhydrogenase present in the 

 mitochondrial membranes of heart muscle 

 was shown by Ball and Cooper (1957) to be 

 inhibited by 4 X 10"^ m thyroxine. The 

 estrogen-sensitive transhydrogenase of the 

 placenta is also inhibited by thyroxine (Vil- 

 lee, 1958b). The degree of inhibition is a 

 function of the concentration of the thyrox- 

 ine and the inhibition can be overcome by 

 increased amounts of estrogen. Suitable con- 

 trol experiments show that thyroxine at 

 this concentration does not inhibit the glu- 

 cose 6-phosphate dehydrogenase or isocitric 

 dehydrogenase used as TPNH-generating 

 systems to couple with the transhydro- 

 genase. Triiodothyronine also inhibits the 

 estrogen-sensitive transhydrogenase but 

 tyrosine, diiodotyrosine and thyronine do 

 not. The thyroxine does not seem to be 

 inhibiting by binding the divalent cation, 

 Mn + + or ]Mg+ + , required for activity, for 

 the inhibition is not overcome by increasing 

 the concentration of the cation 10-fold. 



In the intact animal estrogens stimulate 

 the growth of the tissues of certain target 

 organs. The estrogen-sensitive enzyme has 

 been shown to be present in many of the 

 target organs of estrogens: in human endo- 

 metrium, myometrium, placenta, mammary 

 gland, and mammary carcinoma, in rat ven- 

 tral prostate gland and uterus, and in mam- 

 motrophic-dependent transplantable tumors 

 of the rat and mouse pituitary. In contrast, 

 it is not demonstrable in comparable prepa- 

 rations from liver, heart, lung, brain, or 



