Table 2*. Oxidation Of Estradiol 17β By Ammonium Sulfate Of Placental Fractions

* The activities were measured by observing the reduction of pyridine nucleotide at 340 mµ in systems of 3.0 ml. volume containing 300 imoles Tris pH 7.4; 1 jimole DPN or TPN; 0.074 pinole estradiol 17β in 0.01 ml. dioxane, and appropriate amounts of ensyme. Readings were made at intervals against control cuvettes containing all ingredients except estradiol 170. Temperature 25°.

Inhibition By Mercuric Ions

Low concentrations of mercuric ions inhibited the enzymatic reduction of TPN and DPN by estradiol 17β. The degree of inhibition of the transhydrogenation reaction by Hg++ was found to be of the same order of magnitude. The latter reaction was carried out with a sufficient excess of added isocitric dehydrogenase. Whereas 2 X 10-5 M HgCl2 had little effect on both reactions, brief preincubation of the enzyme with 4 X 10-' M HgCl2 inhibited both the hydroxysteroid dehydrogenase and the transhydrogenating reactions by 80-90 per cent. The placental and certain other hydroxysteroid dehydrogenases are known to be inactivated by heavy-metal ions and have been presumed to be sulf-hydryl group containing proteins.4,8,10

Discussion

It is evident from these experiments that the increased rate of reduction of DPN promoted by steroid hormones in crude extracts of placenta is explicable in terms of an activation of the transfer of hydrogen from TPNH to DPN by steroids. Thus it is not necessary, as others have,2, 3 to postulate the separate existence of a DPN-specific isocitric dehydrogenase as responsible for these hormonal effects.

There is considerable evidence that DPN and TPN serve different metabolic functions and that the natural occurrence of these two different pyridine nucleotides is of profound functional significance. TPNH can act as a reducing agent in a number of synthetic reactions which take place outside the mitochondria and in which DPNH cannot participate. Examples of such biosynthetic pathways are (a) the synthesis of fatty acids, where TPNH acts as a specific reductant in fatty acyl dehydrogenase reactions,11- 11 and (6) the entry of one-carbon fragments into serine13 and into purines14 catalyzed by a series of folic acid-dependent enzyme systems which utilize TPNH as a specific hydrogen donor.16

It is indeed remarkable that studies on the metabolic concomitants of the action of steroid hormones, upon accessory sexual tissues16 have shown that these same extra-mitochondrial synthetic reactions, which specifically require TPNH, are extremely sensitive to the action of steroids. Thus Mueller16 found that, shortly after the administration of estradiol 17β to the ovariectomized rat, the incorporation of one-carbon fragments (derived from a variety of precursors) into serine and into the purines of nucleic acids in the uterus was increased immensely. In similar experiments, estradiol 17/3 stimulated the conversion of acetate to fatty acids and to cholesterol but did not influence the oxidation of acetate.

Furthermore, a number of biochemical changes in the accessory organs of reproduction of the male induced by testosterone could have, as their common denominator, a change in the balance between TPNH and DPNH. Androgenic steroids initiate and support the accumulation and secretion of fructose and citric acid in some male accessory sexual tissues.17 The synthesis of fructose by these organs involves the reduction of glucose to sorbitol by TPNH, followed by the DPN-linked oxidation of sorbitol to fructose.18-19 The over-all conversion of glucose to fructose thus simulates the action of pyridine nucleotide transhydrogenase insofar as there is a stoichiometric transfer of hydrogen from TPNH to DPN in this process. Those lobes of the prostate gland which accumulate and secrete citrate in response to steroids do, in fact, possess the enzymatic machinery for the oxidation of citrate by the tricarboxylic acid cycle, involving the action of a TPN-specific isocitric dehydrogenase.10 Citrate-and no other organic acid-probably accumulates in response to steroids because the unfavorable TPNH/TPN ratio acts to brake the isocitric dehydrogenase reaction and prevents the rate of oxidation of citrate from keeping pace with its rate of synthesis.21 Again, the synthesis of fatty acids from acetate in the prostate gland is most sensitive to testosterone."

In all mammalian tissues examined, including accessory sexual organs, TPNH is present in far higher concentrations than TPN, whereas the steady-state level of DPN is usually greater than that of DPNH." Hence the ratio is always very high. This quotient is the equilibrium constant of the pyridine nucleotide transhydrogenase reaction. The vast majority of pyridine nucleotide-linked dehydrogenases employ only one type of hydrogen acceptor; they do not function equally well with TPN and DPN. Any disturbance in the balance between TPNH and DPNH by a transhydrogenating mechanism would alter the nice equilibrium between synthesis and degradation required for the orderly growth and function of cells. Since there is not a uniform intracellular distribution of pyridine nucleotides and of enzymes with which they react, the factors which determine the steady-state levels of the reduced and oxidized forms of these coenzymes will vary in different regions of the cell.11 Similar considerations concerning the regulation of the balance between hydrogenation and phosphorylation have been advanced by Hoch and Lipmann.24

The existence of enzyme systems which catalyze the transfer of hydrogen between the two natural forms of pyridine nucleotide is well documented. Kaplan et erf." have purified a soluble transhydrogenase, apparently a discrete protein, from Psetidomonas fluorescens. In mammalian species, the transfer of hydrogen between the oxidized and reduced forms of TPN and DPN, between certain unnatural pyridine nucleotides, and even from DPNH to DPN has been observed heretofore only with mitochondrial preparations from some, but not all, tissues.26, 27 The mitochondrial transhydrogenase system has not been purified extensively. It is by no means clear whether the hydrogen transfer from one pyridine nucleotide to another carried out by mitochondria is catalyzed by a single enzyme or reflects the operation of a coupled reaction, whereby an enzyme with dual nucleotide specificity alternately oxidizes and reduces an intermediate, present in minute amounts and thereby permits a rapid net transfer of hydrogen from one pyridine nucleotide to another.

Kaplan28 has suggested that the mitochondrial pyridine nucleotide transhydrogenase system plays an important role in regulating the balance between various synthetic and energy-yielding reactions. His experiments suggest that, while the oxidation of DPNH by liver mitochondria serves as a source of energy for the synthesis of adenosine triphosphate, the oxidation of TPNH is not coupled with phosphorylation unless hydrogen is first donated from TPNH to DPN by the mitochondrial transhydrogenase reaction.

In our studies, those steroid hormones which accelerate the transfer of hydrogen between two forms of pyridine nucleotide also reduce both DPN and TPN in the presence of the same soluble placental enzymes. The enzyme activities are associated in certain protein fractions and absent from others. Both activities are inhibited by similar concentrations of Hg++ to approximately the same extent. Furthermore, the activities are of comparable magnitude in the preparations studied. The simplest interpretation of these experimental findings is that the reversible oxidation and reduction of the steroids themselves constitute the steroid-activated transhydrogenating mechanism. Consider, for example, the case of estradiol 17β. The rapid conversion of this steroid to estrone with either TPN or DPN as a hydrogen acceptor would establish an equilibrium mixture of estrone and estradiol 17β. We visualize the transfer of hydrogen from TPNH to DPN as taking place according to the following equations: