A new hypothesis based on suicide substrate inhibitor studies for the mechanism of action of aromatase. (17/20)

Recently, it was discovered that 4-hydroxy-4-androstene-3,17-dione, 4-androstene-3,6,17-trione, and 1,4,6-androstatriene-3,17-dione, compounds previously reported to be competitive inhibitors of aromatase, cause a time-dependent loss of aromatase activity in human placental microsomes. We report here that 1,4-androstadiene 3,17-dione (Ki 0.32 microM; kinact 0.91 X 10(-3)/sec) and testolactone (Ki 35 microM; kinact 0.36 X 10(-3)/sec) also cause a similar loss of aromatase activity. The mechanism which explains the unexpected loss of activity caused by these five inhibitors is neither established nor apparent from current theories of the enzyme mechanism of action of aromatase. We propose an inactivation mechanism based on a new hypothesis for estrogen biosynthesis in which the third enzyme oxidation carried out by aromatase results in the formation of an enzyme-bound intermediate. This intermediate is released as an aromatized product via a facile elimination reaction which simultaneously regenerates the unaltered active enzyme. Various structural modifications made in these five inhibitors are hypothesized to redirect this elimination reaction so that the steroid intermediate remains covalently attached to the enzyme instead of being released as an aromatized product.  (+info)

Is estrogen required for implantation in the ferret? (18/20)

A series of experiments was designed to further test the hypothesis that ferret corpora lutea synthesize and secrete estrogen between Days 6 and 8 of pregnancy, and that this estrogen is required to initiate implantation of blastocysts on Day 12. Corpora lutea, removed on Day 8 of pregnancy contained significant quantities of testosterone. Incubation of aliquots of the same luteal tissue for 4 h significantly elevated estrogen levels above those of nonincubated controls. Peripheral plasma estrogen levels exhibited a slight increase on Day 8 over those observed on Day 6 of pregnancy (7.9 +/- 3.4 vs. 4.1 +/- 1.1 pg/ml). However, continuous release of estradiol from Days 6-8 from two different sizes of Silastic capsules failed to induce implantation in ovariectomized-progesterone treated ferrets, whereas this same treatment was compatible with nidation in intact ferrets. Administration of the aromatase inhibitor, androsta-1,4,6-triene-3,17-dione (ATD) on Days 5-8 of pregnancy prevented implantation of blastocysts on Day 13. Simultaneous administration of estradiol and ATD failed to reverse the inhibitory effect of ATD. Results of this study confirm that luteal tissue of ferrets possesses at least one aromatizable androgen which is converted to estrogen under physiological conditions. However the data do not support the hypothesis that estrogen is required for implantation in the ferret.  (+info)

Active site-directed photoinactivation of delta 5-3-ketosteroid isomerase from Pseudomonas putida dependent on 1,4,6-androstatrien-3-one-17 beta-ol. (19/20)

delta 5-3-Ketosteroid isomerase from Pseudomonas putida is subject to photoinactivation by light of wavelengths greater than 300 nm, specifically in the presence of the competitive inhibitor, 1,4,6-androstatrien-3-one-17 beta-ol (TEO). In the absence of this steroid or in the presence of the nonchromophoric steroidal competitive inhibitor, deoxycholate, the enzyme activity is essentially unaffected by irradiation. Deoxycholate protects the enzyme from the TEO-dependent reaction to a degree which is predictable from the concentrations of deoxycholate and TEO and their respective competitive inhibition constants, thus demonstrating that the inititial velocity of the photoinactivation is dependent upon the fraction of enzyme active sites occupied by TEO. Cholate, which is not a competitive inhibitor, does not protect enzyme activity. Amino acid analyses of hydrochloric acid hydrolysates of the photoinactivated enzyme show no significant differences from that of the native enzyme. However, the fraction of initial enzyme activity remaining correlates quantitatively with the disappearance of one of the four thiol groups in each polypeptide chain of the enzyme. Enzyme irradiated under the same conditions in the absence of TEO does not lose thiol groups.  (+info)

Prenatal inhibition of aromatase activity affects luteinizing hormone feedback mechanisms and reproductive behaviors of adult guinea pigs. (20/20)

The necessity of brain aromatization for sexual differentiation was investigated by treating pregnant guinea pigs with an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD), from Day 30 to Day 55 of gestation. In postnatal Week 16, subjects were gonadectomized, and blood samples were collected after treatment with 10 micrograms estradiol benzoate (EB), used to elicit an LH surge; subjects were subsequently treated with GnRH to test pituitary responsiveness. Plasma samples were assayed for LH by RIA. Prenatal ATD treatment did not affect the organization of the LH surge mechanism in either male or female subjects. All animals, regardless of sex or treatment, released LH after GnRH treatment, but the responsiveness of the gonadotroph to GnRH was attenuated in both males and females treated with ATD in utero. In addition, a significant sex difference in the pattern of LH released in response to a GnRH challenge was found. ATD-treated animals did not respond to the negative feedback actions of EB on LH secretion (p < 0.05), and the percentage of males displaying lordosis behavior was greater in this group than in controls (p < 0.05). Mounting behavior and lordosis behavior of females were not significantly affected by treatment. These data demonstrate a need for estrogen in the organization of brain areas that mediate negative feedback control of LH in both sexes and lordosis behavior in the male guinea pig. The organization of positive feedback mechanisms for controlling LH seems to be under androgenic control. Our data also suggest that the responsiveness of the gonadotroph to GnRH action is developmentally coordinated by prenatal estrogen and is sexually differentiated.  (+info)