It will be shown in this paper that a soluble enzyme from human placenta promotes the transfer of hydrogen between two pyridine nucleotide coenzymes and that this reaction is activated by minute quantities of certain steroid hormones. This enzyme system catalyzes the over-all reaction:

TPNH + DPN+ → TPN+ + DPNH, where DPN and TPN refer to the oxidized forms of di- and triphosphopyridine nucleotides and DPNH and TPNH designate the respective reduced nucleotides. Consideration will be given to the pivotal role of this reaction in the control of metabolic processes. The implications of the control of pyridine nucleotide balance by steroids will be discussed in relation to the physiological actions of these hormones.

Recent experiments of Villee et al.1, 1 showed that, in the presence of isocitrate the reduction of DPN catalyzed by crude extracts of placenta was accelerated by certain steroids. These findings were interpreted in terms of a steroid activation of a DPN-specific isocitric dehydrogenase. The observations of Villee were readily confirmed in this laboratory. However, it was found that the response of crude placental extracts to steroids was variable and that the remarkable stimulation of the reduction of DPN by certain hormones disappeared upon fractionation of the extracts. We have observed that the addition of catalytic quantities of TPN to the reaction mixture increased markedly the ability of different placental preparations to respond to steroids and restored this effect in preparations which were inactive in this respect. It will be shown that the apparent stimulation of a DPN-linked isocitric dehydrogenase can be accounted for in terms of a coupling of the TPN-specific isocitric dehydrogenase of placenta with the action of a soluble trans-hydrogenating system here described:

Only step 6, the transhydrogenase system, is activated by steroids. Isocitric dehydrogenase is not the only TPN-specific enzyme which can act as a generator for TPNH in this coupled reaction. It can, for instance, be replaced equally well by glucose-6-phosphate dehydrogenase.

Partially purified fractions from placenta which catalyze hydrogen transfer between the two forms of pyridine nucleotide also catalyze the oxidation of steroids by DPN and TPN. Of the steroids examined, only those which can undergo oxido-reduction by pyridine nucleotide-linked hydroxysteroid dehydrogenases4 are capable of activating the transhydrogenase mechanism. The experiments are consistent with the view that the proteins catalyzing dehydrogenation and transhydrogen-ation are identical.

By Paul Talalayj and H. G. Williams-Ashman

Ben May Laboratory For Cancer Research And Department Of Biochemistry, University Of Chicago

Communicated by Charles Hoggins, November 7, 1967

Methods

Preparation Of Placental Enzymes

Crude extracts were prepared by the method of Villee and Gordon.2,3 Human placenta was obtained immediately after birth and placed on ice. As much blood as possible was removed by perfusion with approximately 1 liter of isotonic sodium chloride. The tissue was homogenized with 4-5 volumes of ice-cold 0.25 M sucrose, either in a glass homogenizer equipped with a Teflon pestle or in a Waring Blendor. All further operations were carried out at less than 2°. The homogenates were centrifuged at 2,000 X g for 15 minutes, and the resulting supernatant fluid was centrifuged at 59,000 X g for 1 hour in a Spinco preparative ultracentrifuge. The supernatant fluid obtained from the second cen-trifugation was stored either in an ice-bath or in the frozen state and will be referred to as the "crude placental extract." Fractionation was carried out with ammonium sulfate at 0°. The precipitates which formed upon the addition of various amounts of ammonium sulfate were collected by centrifugation and were dissolved in 0.01 M tris(hydroxymethyl)aminoethane (Tris) buffer of pH 7.4. These fractions were stored at -10°.

Determinations

Spectrophotometric measurements were made with a Beckman model DU spectrophotometer using either Pyrex or silica cells of 1 cm. light path. The formation of reduced pyridine nucleotides was followed by measurement of change in absorbance at 340 mµ. The molar extinction coefficient of reduced pyridine nucleotides at 340 mu was assumed to be 6,220. The TPN was estimated with purified isocitric dehydrogenase of rat heart in Tris buffer at pH 9.0 in the presence of an excess of isocitrate and 0.0003 M MnCl2. The isocitrate was determined in the same system with an excess of TPN. Glucose-6-phosphate and DPN were estimated with the aid of yeast glucose-6-phosphate and alcohol dehydrogenases, respectively. Protein was determined by the method of Warburg and Christian.5

Materials

DPN and TPN were commercial products of greater than 85 per cent purity and were uncontaminated by each other. Isocitric dehydrogenase, purified from rat heart by a slight modification of the method of Siebert et al.,' was generously donated by S. K. Wolfson, Jr. This purified enzyme did not reduce DPN upon the addition of isocitrate and MnCU in the presence or absence of estradiol 17/3. It had a specific activity of 0.6 jumole of isocitrate oxidized per minute per milligram protein under the conditions of the experiments to be described. Twice recrystallized yeast alcohol dehydrogenase was obtained as a paste suspended in 60 per cent saturated ammonium sulfate solution from the Sigma Chemical Company, St. Louis, Missouri. It was diluted with ice-cold deionized water just prior to use. The samples of estradiol 17/3 and of testosterone were of greater than 98 per cent purity as determined by enzymatic assay.7 The other steroids and di-ethylstilbestrol were of high purity. All other preparations were of commercial origin.

Results

Response of Crude Extracts of Placenta to Estradiol 170.-The experiments were carried out under conditions slightly different from those described by Gordon and Villee. 2,3 Each vessel contained 300 µmoles Tris buffer of pH 7.4,1 µmole of DPN, and 1.0 ml. of a crude extract of placenta equivalent to approximately 0.2 gm. of fresh tissue. The total volume was made up to 3.0 ml. with water, and estradiol 17/3 was added in 0.01 ml. of dioxane, which was also added to the control vessels.

The reduction of DPN by some crude placental extracts was accelerated markedly by estradiol 17/3 at a final concentration of 5 X 10^-6 M, with or without the addition of isocitrate (1 X 10~3 M) or glucose-6-phosphate (1 X 10^-3 M) to the reaction mixture. Under similar conditions, other placental preparations did not respond to estradiol 17/3, but it was possible to restore their sensitivity to this hormone by the addition of catalytic amounts (0.01-0.05 ^mole) of TPN.

Further experiments with crude placental extracts revealed that (a) these preparations reduced TPN very rapidly in the absence or presence of added substrates (the reduction of TPN was unaffected by estradiol 17β); (b) the magnitude of the increase in rate of DPN reduction induced by estradiol 17/3 was enhanced by the addition of TPN-reducing substrates, such as isocitrate or glucose-6-phosphate; and (c) even with those extracts of placenta in which the steroids accelerated the reduction of DPN in the absence of exogenous TPN, the addition of catalytic amounts of the latter nucleotide magnified the stimulation induced by estradiol 170.

These phenomena are explicable in terms of a coupled reaction between a TPN-specific isocitric dehydrogenase or a TPN-specific glucose-6-phosphate dehydrogenase and an enzymatic mechanism which could transfer hydrogen from TPNH to DPN (see eqs. [a] and [6]). Presumably those extracts which were stimulated by estradiol 17/3 without the further addition of catalytic amounts of TPN contained sufficient quantities of the latter nucleotide to allow the coupled reaction to proceed. The amount of TPN required was found to be very small, since as little as 0.01 µmole of TPN restored the ability of inactive placental extracts to respond to estradiol 17/3. Definitive evidence in support of this hypothesis required enzyme preparations free from endogenous substrates and TPN. Extracts of acetone powders of the placental enzymes were not suitable, but fractionation with ammonium sulfate provided a simple method for removing interfering substances.