TRANSHYDROGENASE REACTIONS AND ESTROGENS 173 



genases, specific for DPN and TPN, respectively. Hagerman and 

 Villee (1959) described the separation of these enzymes by elec- 

 trophoresis on a starch l^lock and also bv filter-paper curtain electro- 

 plioresis in bar])ital buffer of pH 8.6. The transhvdrogenase and the 

 two estradiol dehydrogenases were also separated by chromatography 

 on DEAE cellulose (Villee, Hagerman, and Joel, 1960). The speci- 

 ficity of the two estradiol dehydrogenases toward various pyridine 

 nucleotide analogues, or toward different steroids, was not reported. 

 Hagerman and Villee ( 1959 ) found that a mixture of their placental 

 DPN- and TPN-linked estradiol dehxxlrog-enases did not catalyze 

 the transfer of h\ drogen from TPNH to DPN in the presence of es- 

 tradiol-17^. Thev put forward the hypothesis that estradiol-17^ 

 stimulates the TPNH-DPN and the DPNH-acetylpyridineDPN trans- 

 hydrogenase reactions by converting, in an unspecified manner, an 

 inactive form of the placental enzyme into an active one. This im- 

 plies that estradiol-17yS or estrone does not undergo alternate oxida- 

 tion and reduction during the course of these transhydrogenations 

 and that the steroids cannot be regarded as hydrogen carriers in 

 these reactions.^ 



Binding and Interactions of Pyridine Nucleotides 



The affinity of the purified placental enzyme for various pyridine 

 nucleotides in the dehydrogenase reaction with estradiol- 17/3 and 

 estrone as substrates has been studied extensively ( Talalay, Hurlock, 

 and Williams-Ashman, 1958; Talalav and Williams-Ashman, 1960). 

 At pU 7.4, the Michaelis constant for TPNH^TPN<DPNH<DPN< 

 acetvlp\ridineDPN. The affinity for TPN(H) was so high that their 

 Michaelis constants could not be measured accurately by the usual 

 spectrophotometric procedures, and the same was true for acetyl- 

 pvridineTPN. The affinity for acetvlpyridineDPN and pyridinealde- 

 hvdeDPN is relatively low, and the rates of oxidation of estradiol-17yS 

 by the latter nucleotides are unusually sensitive to changes in pH. 



The reduction of DPN bv estradiol-17/3 was inhibited almost com- 

 pletely by one-hundredth the molar concentration of TPN, and de- 



3 Note added in proof. Since this article was submitted for publication, Drs. J. 

 Jarabak and P. Talalay in this laboratory ha\e purified the placental hydroxysteroid 

 dehydrogenase more than 2500-fold, and in high yield. No separation between various 

 dehydrogenase and transhydrogenase functions was apparent at any stage of the 

 purification procedure whicli involved, inter alia, chromatography on ion-exchange 

 resins and electrophoresis. 



