TRANSHYDROGENASE REACTIONS AND ESTROGENS 175 



in situ by the action of alcoliol deliydrogeiiase) to TPN in tlie absence 

 or presence of estradiol-17^. Villee and Hagernian (1958) reported 

 similar findings and conclnded that the estradiol TPNH-DPN trans- 

 fer was irreversible. However, Talalay, Hurlock, and Williams-Ash- 

 man (1958) demonstrated tliat this reaction was readily reversible 

 under appropriate experimental conditions, namely, with a high 

 initial ratio of DPNH to TPN at /}H 7.4. The reaction was followed 

 by measuring the disappearance of DPNH (IXIQ-^M) in the 

 presence of low levels of TPN and a specific TPNH-oxidizing system 

 (oxidized glutathione and glutathione reductase): 



DPNH + TPN+ ^ DPN+ + TPNH 

 H+ + TPNH + GSSG-^ 2 GSH + TPN + 



H+ + DPNH + esse -^ 2 GSH + DPN + 



The optimal concentration of TPN was found to be 1.6 X lO^*^ M. 

 Marked inhibition of the DPNH-TPN reaction was observed if the 

 TPN level was raised to 5.4 X 10 ~*^ M, and almost complete inhibi- 

 tion occurred when the TPN concentration was 1.6 X 10~' M. This 

 situation is reminiscent of the inhibition by TPN of the TPNH-DPN 

 exchange in the forward direction and also of the transfer of hydrogen 

 between DPNH and acetylpvridineDPN. Very recently Hosoya, 

 Hagerman, and Villee (1960) have confirmed the reversibility of 

 the estradiol-mediated TPNH-DPN transhydrogenase reaction. 



Steroid Specificity 



The specificity for steroids in both the dehydrogenase and the 

 transhvdroeenase reactions is verv similar. Hollander, Nolan, and 

 Hollander (1958) showed that of some 30 substituted 1, 3, 5-estra- 

 triene derivatives, only those compounds bearing a 17yS-hydroxyl or 

 17-ketone group stimulated the transfer of hydrogen from TPNH to 

 DPN by crnde placental extracts. In a series of synthetic racemic 

 isoestrones, onlv <://-8-i5oestrone functioned as a mediator of the trans- 

 hydrogenase reaction and as a substrate in the dehydrogenase re- 

 action (Hollander, Hollander, and Brown, 1959b). The steroid 

 specificity for the dehvdrogenase reaction with DPN as hydrogen 

 acceptor has been stndied exhanstively by Langer, Alexander, and 

 Engel (1959). They found onl\' three compounds which failed to 

 function in both the dehydrogenase and the transhydrogenase reac- 



