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A High Affinity Inhibitor of Pituitary Progesterone 5-Reductase^*

Department of Physiological Chemistry and The Waisman Center on Mental Retardation and Human Development, The University of Wisconsin-Madison Madison, Wisconsin 53706

Address requests for reprints to: Dr. Harry J. Karavolas, Department of Physiological Chemistry, Center for Health Sciences, 1300 University Avenue, 589 Medical Sciences Building, University of Wisconsin-Madison, Madison, Wisconsin 53706.

Abstract

The capacity of the 5a-dihydroprogesterone analog, 4-aza-4-methyl-5-pregnane-3,20-dione (AMPD), to inhibit progesterone 5-reductase and both 5-dihydroprogesterone 3-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-reduction but is ineffective at inhibiting either of the 3-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 µM. A kinetic analysis of the interaction of AMPD with progesterone 5-reductase, indicates that it is a competitive inhibitor vs. progesterone [K_ialope = 7.2 ± 0.6 nM; apparent Michaelis-Menten constant (K_m) (progesterone) = 193 ± 18 nM] and an uncompetitive inhibitor vs. NADPH (K_iintercept = 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-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-reductase and the function of anterior pituitary progestin metabolism. (Endocrinology 114: 63, 1984)

Footnotes

^* This investigation was supported by USPHS Grants 1-R01-HD-05414, 5-T32-HD-07007, 5-T32-GM-07215, and Ford Foundation Grant 630-0505B,C. A preliminary report of these findings was presented at the 65th Annual Meeting of The Endocrine Society, San Antonio, Texas, 1983.

Received April 14, 1983.