STEROID SEX HORMONES 



655 



nents of the cell. This would occur, of course, 

 only if the supply of ATP, rather than the 

 amount of enzyme, substrate, or some other 

 cofactor, were the rate-limiting factor in the 

 synthetic processes. 



When purified estrogens became avail- 

 able, they were tested for their effects on 

 tissues in vitro. Estrogens added in vitro in- 

 creased the utilization of oxygen by the rat 

 uterus (Khayyal and Scott, 1931) and the 

 rat pituitary (Victor and Andersen, 1937). 

 The addition of estradiol- 17^ at a level of 

 1 fxg. per ml. of incubation medium increased 

 the rate of utilization of oxygen and of 

 pyruvic acid by slices of human endome- 

 trium and increased the rate at which la- 

 beled glucose and pyruvate were oxidized to 

 C^-^Os (Hagerman and Villee, 1952, 1953a, 

 1953b) . In experiments with slices of human 

 placenta similar results were obtained and 

 it was found that estradiol increased the 

 rate of conversion of both pyruvate-2-C^'* 

 and acetate-l-Ci4 to C^^Os (Villee and 

 Hagerman, 1953) . From this and other evi- 

 dence it was inferred that the estrogen acted 

 at some point in the oxidative pathway 

 common to pyruvate and acetate, i.e., in the 

 tricarboxylic acid cycle. 



Homogenates of placenta also respond to 

 estradiol added in vitro. With citric acid as 

 substrate, the utilization of citric acid and 

 oxygen and the production of a-ketoglutaric 

 acid were increased 50 per cent by the 

 addition of estradiol to a final concentra- 

 lion of 1 fjig. per ml. (Villee and Hagerman, 

 1953). The homogenates were separated by 

 differential ultracentrifugation into nuclear, 

 mitochondrial, microsomal, and nonparticu- 

 late fractions. The estrogen-stimulable sys- 

 tem was shown to be in the nonparticulate 

 fraction, the material which is not sedi- 

 mented by centrifugating at 57,000 X g 

 for 60 minutes (Villee, 1955). Experiments 

 with citric, as-aconitic, isocitric, oxalosuc- 

 cinic, and a-ketoglutaric acids as sub- 

 strates and with fluorocitric and trans- 

 aconitic acids as inhibitors localized the 

 estrogen-sensitive system at the oxidation 

 of isocitric to oxalosuccinic acid, which then 

 undergoes spontaneous decarboxylation to 

 a-ketoglutaric acid (Villee and Gordon, 

 1955). Further investigations using the en- 

 zymes of the nonparticulate fraction of 

 the human placenta revealed that, in ad- 



dition to isocitric acid as substrate, only 

 DPN and a divalent cation such as Mg+ + 

 or Mn++ were required (Villee, 1955; Gor- 

 don and Villee, 1955; Villee and Gordon, 

 1956). The estrogen-sensitive reaction was 

 formulated as a DPN-linked isocitric de- 

 hydrogenase: 



Isocitrate + DPN* -^ a-ketoglutarate 



+ CO2 + DPXH + H* 



It was found that the effect of the hor- 

 mone on the enzyme can be measured by 

 the increased rate of disappearance of citric 

 acid, the increased rate of appearance of 

 a-ketoglutaric acid, or by the increased 

 rate of reduction of DPN, measured spectro- 

 photometrically by the optical density at 

 340 m/x. As little as 0.001 /xg. estradiol per 

 ml. (4 X 10~^ m) produced a measurable 

 increase in the rate of the reaction, and 

 there was a graded response to increasing 

 concentrations of estrogen. The dose-re- 

 sponse curve is typically sigmoid. This sys- 

 tem has been used to assay the estrogen 

 content of extracts of urine (Gordon and 

 Villee, 1956) and of tissues (Hagerman, 

 Wellington and Villee, 1957; Loring and 

 Villee, 1957). 



Attempts to isolate and purify the estro- 

 gen-sensitive enzyme were not very success- 

 ful. By a combination of low temperature 

 alcohol fractionation and elution from cal- 

 cium phosphate gel a 20-fold purification 

 was obtained (Hagerman and Villee, 1957). 

 However, as the enzyme was purified it was 

 found that an additional cofactor was re- 

 quired. Either uridine triphosphate (UTP) 

 or ATP added to the system greatly 

 increased the magnitude of the estrogen ef- 

 fect and, subsequently, adenosine diphos- 

 phate (ADP) was recovered from the in- 

 cubation medium and identified by paper 

 chromatography (Villee and Hagerman, 

 1957). Talalay and Williams-Ashman 

 (1958) confirmed our observations and 

 showed that the additional cofactor was 

 triphosphopyridine nucleotide (TPN) which 

 was required in minute amounts. This find- 

 ing was confirmed by Villee and Hagerman 

 (1958) and the estrogen-sensitive enzyme 

 system of the placenta is now believed to be 

 a transhydrogenase which catalyzes the 

 transfer of hydrogen ions and electrons 



