TRANSHYDROGENASE REACTIONS AND ESTROGENS 169 



bound xery firmh' to tliis enzyme system, with a dissociation con- 

 stant of 2.6 X 10~^^ M. \'illee concluded from this fact that the hor- 

 mone combined witli an inactive form of the enzyme and thereby 

 converted the protein into an enzymaticall)- active form. 



Transhydrogenase Reaction 



The enhancement by estradiol-17;S of DPNH formation in such 

 crude placental extracts is variable and usually disappears upon frac- 

 tionation of the extracts by treatment with ammonium sulfate. But 

 with fractionated enz' me preparations, activity could be restored 

 by the addition of catalytic amounts of TPN (Talala\' and Williams- 

 Ashman, 1958). This suggested that estradiol-17yS did not activate 

 a DPN-linked isocitric deh^xlroo■enase but stimulated the transfer of 

 hydrogen from TPNH to DPN. The apparent activation of a DPN- 

 specific isocitric dehydrogenase could be accounted for by the fol- 

 lowing coupled reaction: 



Isocitrate + TPN+ ^ a-Ketoglutarate + COo + TPNH 

 TPNH + DPN+ ^ TPN+ + DPNH 



Isocitrate + DPN+ -> a-Ketoglutarate + COo + DPNH 



This formulation implies that the action of estradiol- 17^ promoted 

 the transfer of hydrogen from one form of pyridine nucleotide to 

 another and was in no special wav connected with the oxidation of 

 isocitrate. It was supported by the demonstration ( Talalay and Wil- 

 liams-Ashman, 1958; Talalay, Hurlock, and Williams-Ashman, 1958) 

 that TPN-specific dehydrogenases for either glucose-6-phosphate or 

 acetaldelnde could replace isocitric dehydrogenase as a TPNH- 

 generating system. Moreover, an estradiol-dependent transfer of 

 hydrogen to DPN from TPNH added as such to the reaction mix- 

 ture was demonstrable in the presence of low levels of DPN and a 

 specific DPNH-oxidizing system ( acetaldehyde and yeast alcohol 

 dell vdrogenase ) . 



Villee and Hagerman (1958) and Hollander, Hollander, and 

 Brown (1959a) confirmed the requirement for catalytic levels of 

 TPN and a TPNH-reducing system for the estradiol-mediated re- 

 duction of DPN by crude placental extracts. The hypothesis that 

 estradiol activated a DPN-specific isocitric dehydrogenase was re- 

 tracted by Villee and Hagerman (1958), who accepted the conclu- 



