This oxidase system was bound to cytoplasmic particles and appeared to have many properties in common with the uterine peroxidase described by Stotz et al. (80, 84). Both activities were negligible in extracts of the uteri of spayed animals, but became apparent shortly after the injection of either natural or synthetic estrogens. Bever et al. (13) found that the administration of estrogen increased the DPNH oxidase levels of the uteri of spayed rats.

We have confirmed and extended (79) the observations of Hollander and Stephens. In our experience, 3,4-dimethylphenol was a particularly active mediator for the oxidation of DPNH, as shown in Table IX. This reaction is inhibited by cyanide and by catalase. The enzyme system is bound to cytoplasmic particles which are sedimented from homogenates prepared in 0.25 M sucrose by centrifugation between 600 and 14,000 g. The enzyme system can be removed from these particles by extraction with 1.2 M NaCl and has been purified about threefold by fractionation with ammonium sulfate. Table X shows the influence of the administration of diethylstilbestrol on the activity of a number of oxidizing enzymes associated with cytoplasmic particles of the uteri of spayed rats. Estrogen administration increased the DPNH-cytochrome c reductase and also the rate of aerobic oxidation of DPNH in the presence of cytochrome c, approximately twofold. But the rates of the phenol-stimulated oxidation of DPNH, and of the uterine peroxidase (oxidation of a leucoindophenol by hydrogen peroxide) were increased 142- and 65-fold, respectively. It can be seen that with uterine particles derived from spayed animals treated with diethylstilbestrol, the rate of aerobic oxidation of DPNH in the presence of 3,4-dimethylphenol and Mn++ was about a hundred times that observed with cytochrome c as the added carrier.

Table IX. Stimulation By 3,4-Dimethylphenol Of The Aerobic Oxidation Of Dpnh Catalyzed By An Extract Of Uterine Cytoplasmic Particles*

System

ΔE340 per 10 min.

Complete

0.172

Omit 3,4-dimethylphenol

0.000

Omit MnCl2

0.017

Omit enzyme

0.019

* Reactions carried out at 25° C. in a final volume of 3.0 ml., containing 250 umoles Tris buffer pH 7.4; 0.3 umole DPNH; 1 µmole MnCl2 and 10 umoles 3,4-dimethyl-phenol. The enzyme was prepared from cytoplasmic particles (isolated from a 0.25 M sucrose homogenate) by extraction with 1.2 M NaCl and was equivalent to 0.5 mg. of fresh tissue.

Very recently, Temple and Hollander (118) reported that certain steroidal estrogens stimulated the oxidation of DPNH by uterine particles incubated with Mn++. The optimal pH for this estradiol-mediated reaction was considerably higher than that observed when the estrogen was replaced by 2,4-dichlorophenol. This action of estradiol-17β in vitro is remarkably similar to that found in the model systems catalyzed by highly purified peroxidases described above. The relationship of this carrier action of estradiol-17β for the oxidation of DPNH by uterine particles to the induction of uterine peroxidase by estrogenic substances in vivo is of great interest. But it should be noted that Stotz and his collaborators (84) have shown that a number of types of hydrogen donor, including ferrocytochrome c, are oxidized by uterine peroxidase in the presence of hydrogen peroxide. The relative rates of oxidation of various donors by the uterine oxidase system in the presence of Mn++ and of estrogenic or other phenols must be studied more exhaustively before any role of this system in the terminal electron transport in the intact uterus can be surmised.4

Table X. Uterine Enzyme Activities After The Administration Of Stilbestrol To Ovariectomized Rats*

µMoles substrate per

: oxidized or redu mg. particle N

Treated with

ced per min. Stilbestrol:

Enzyme system

Untreated

stilbestrol

untreated

(I)

DPNH-phenol-O2

0.183

26.1

142

(II)

Reduced indophenol-H2O2

0.008

0.519

65

(peroxidase)

(III)

DPNH-cytochrome c-O2

0.119

0.213

1.8

(IV)

DPNH-cytochrome c reductase

0.239

0.421

1.7

* Uterine particles were prepared from female animals (220-250 gm.), 10 days after ovariectomy. Stilbestrol (0.05 mg.) was injected every other day for 8 days. Reactions were carried out in a final volume of 3.0 ml. at 25°C. The components of each assay system were as follows: (I) Phenol-stimulated oxidation of DPNH: 250 (imoles Tris buffer pH 7.4; 1 (imole MnCl2; 10 (imoles 3,4-dimethylphenol; 0.3 (imole DPNH. (II) Peroxidase: 250 (imoles sodium phosphate buffer pH 6.25; 1.8 (imoles hydrogen peroxide; 0.5 (imole reduced 2,3',6-trichloroindophenoI. (Ill) Aerobic oxidation of DPNH: 250 (imoles Tris buffer pH 7.4; 0.3 (imole DPNH; 0.01 umole cytochrome c. (IV) DPNH-cytochrome c reductase: 250 umoles Tris buffer pH 8.5; 0.3 umole DPNH; 0.1 (imole cytochrome c; 2 (imoles KCN. The wavelengths at which the reactions were followed were 340 mu for reactions (I) and (III); 645 m(i for reaction (II) and 550 mil for reaction (IV). Each vessel contained appropriate quantities of the suspension of uterine particles, and nonenzymatic blanks were run when required.

4 The discussion for this chapter is included in the discussion following the chapter by Drs. C. A. Villee, D. D. Hagerman, and P. B. Joel.

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