Alpha-reduction Of Progesterone Research Articles

Estrophilic 3α,3β 17β,20α-hydroxysteroid dehydrogenase from rabbit liver—II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.

Inhibition of androgen metabolism in stroma and epithelium of the human benign prostatic hyperplasia by progesterone, estrone, and estradiol.

It has been shown that progesterone, estrone, and estradiol are present in significant amounts in the human benign prostatic hyperplasia (BPH). Therefore it was of interest to determine the inhibitory effect of these steroids on the 5 alpha-reductase and 3 alpha (beta)-hydroxysteroiddehydrogenase (HSDH) activities, the enzymes responsible for the conversion of testosterone to 5 alpha-dihydrotestosterone (DHT), and DHT to 3 alpha (beta)-androstanediols, respectively. The enzyme inhibition was analyzed in vitro by measuring the 5 alpha-reductase in the presence of either progesterone, estrone, or estradiol, using testosterone as substrate. The DHT was used as substrate for HSDH. The metabolites were quantified by t.l.c. The main results were as follows: (1) Concerning 5 alpha-reductase in BPH, the mean inhibitor constants (KI; microM) of progesterone, estrone, and estradiol were 0.11, 15.5, and 5.1, respectively. (2) Analyzing epithelium and stroma of BPH separately, the inhibition of 5 alpha-reductase resulted in nearly identical KI's. (3) Concerning HSDH in BPH, the mean KI's of progesterone, estrone, and estradiol were 169, 63, and 192, respectively. (4) Analyzing epithelium and stroma of BPH separately, the inhibition of HSDH led to nearly identical KI's. (5) The kinetic parameters (KM, Vmax) of the 5 alpha-reduction of progesterone and testosterone were nearly identical. These results led to the suggestion that the endogenous concentrations of progesterone, estrone, and estradiol have no significant inhibitory effect on the 5 alpha-reductase and HSDH in vivo. Furthermore, the nearly identical inhibitor constants found for both enzymes in epithelium and stroma of BPH indicate that in both compartments the 5 alpha-reductase and HSDH are qualitatively identical.

A high affinity inhibitor of pituitary progesterone 5 alpha-reductase.

The capacity of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit progesterone 5 alpha-reductase and both 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADPH- and NADH-linked) from the female rat anterior pituitary has been investigated. Dose response studies demonstrate that AMPD is a powerful inhibitor of pituitary progesterone 5 alpha-reduction but is ineffective at inhibiting either of the 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. A kinetic analysis of the interaction of AMPD with progesterone 5 alpha-reductase indicates that it is a competitive inhibitor vs. progesterone [Kislope = 7.2 +/- 0.6 nM; apparent Michaelis-Menten constant (Km) (progesterone) = 193 +/- 18 nM] and an uncompetitive inhibitor vs. NADPH (Kiintercept = 17.9 +/- 1.4 nM). These inhibition patterns are consistent with the view that NADPH binding precedes that of either AMPD or progesterone. Furthermore, AMPD does not appear to be an irreversible inhibitor since preincubation of the enzyme (at 37 C) with AMPD and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. The inhibition can also be readily removed by dilution, even after a 60-min preincubation with the inhibitor and NADPH. It is postulated that the selective and potent inhibition of the 5 alpha-reduction of progesterone by AMPD may be due to the steroid functioning as a transition state analog. This inhibitor should prove useful in studying the properties of progesterone 5 alpha-reductase and the function of anterior pituitary progestin metabolism.

A unique change of steroid metabolism in rat liver microsomes induced with highly toxic polychlorinated biphenyl(PCB) and polychlorinated dibenzofuran(PCDF).

Pretreatments of rats with the highly toxic compounds such as 3,4,5,3',4'-pentachlorobiphenyl(PenCB), and 2,3,7,8-tetrachloro(TCDF) and 2,3,4,7,8-pentachlorodibenzofuran(PenCDF), which are potent 3-methylcholanthrene(MC)-type inducers, increased selectively 7 alpha-hydroxylation, but strongly suppressed 2 alpha-, 6 beta- and 16 alpha-hydroxylations as well as 5 alpha-reduction of progesterone and testosterone in the liver microsomes. This unique change in the metabolic pattern was accompanied by a marked decrease in total metabolism of both steroids and appeared to correlate apparently with their toxic potency. This kind of change was not shown by pretreatments with not only MC itself but also the phenobarbital-type(2,4,5,2',4',5'-hexachlorobiphenyl) and the mixed type PCBs(Kanechlor 400, a PCB mixture with 48% chlorine content), all of which possess only a low acute toxicity. The metabolic change produced by 3,4,5,3',4'-PenCB, 2,3,7,8-TCDF and 2,3,4,7,8-PenCDF might not be due to their stimulatory or inhibitory effects, because when added to the incubation mixture 3,4,5,3',4'-PenCB did not change the metabolic pattern with MC-microsomes to that with 3,4,5,3',4'-PenCB-microsomes. Furthermore, either 2,3,7,8-TCDF or 2,3,4,7,8-PenCDF gave similar metabolic pattern whereas their residual levels in the liver were greatly different from each other at the time of sacrifice. These results suggest that this kind of unique change of the steroid metabolism produced by highly toxic PCBs and PCDFs may be responsible, at least in part, for the endocrine symptoms caused by these compounds via disturbance of steroid homeostasis.

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.