Screening for factor V Leiden mutation before prescribing combination oral contraceptives.

OBJECTIVE: To evaluate the cost-effectiveness of screening for factor V Leiden mutation in women in the United States who use combination oral contraceptives. DESIGN: Cost-effectiveness analysis. SETTING: A national research reference laboratory, a university medical center, and an academic health center managed care organization. PATIENT(S): Women of reproductive age in the United States. INTERVENTION(S): Baseline risk estimates of venous thromboembolic disease in the general population and in carriers of factor V Leiden mutation were calculated using available data. MAIN OUTCOME MEASURE(S): The number of women who would require factor V Leiden testing and the cost of identifying this cohort to prevent one death caused by venous thromboembolic disease before prescribing combination oral contraceptives. RESULT(S): To prevent one venous thromboembolic death attributable to the use of oral contraceptives in women with factor V Leiden mutation, >92,000 carriers would need to be identified and stopped from using these pills. The estimated charge to prevent this one death would exceed $300 million. If the price of testing were discounted to 34.5% of current charges, the cost still would be between $105 million and $130 million. CONCLUSION(S): Screening for factor V Leiden mutation before prescribing combination oral contraceptives is not a cost-effective use of U.S. health care dollars. The best and most cost-effective screening tool we have is taking a thorough personal and family history related to venous thromboembolic events.

Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain.

Human 3beta-hydroxysteroid dehydrogenase/steroid Delta(5)-Delta(4)-isomerase (3beta-HSD/isomerase) is a bifunctional, single enzyme protein that is membrane-bound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3beta-HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3beta-HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3beta-HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3beta-HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH(2)-terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/quaternary structure of this key steroidogenic enzyme.

Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.

3beta-hydroxysteroid dehydrogenase/steroid delta5-->4-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3beta-HSD/isomerase (monomeric Mr=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3beta-HSD activity, whereas the Km and Vmax values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The Vmax (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean Vmax (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar Vmax values for substrate oxidation by the 3beta-HSD activity. The 3beta-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD+ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD+ was dramatically decreased. Based on these kinetic measurements, His261 appears to be a critical amino acid residue for the 3beta-HSD activity, and Tyr253 or Tyr254 participates in the isomerase activity of human type I (placental) enzyme.

Affinity radiolabeling identifies peptides associated with the isomerase activity of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.

3beta-Hydroxysteroid dehydrogenase and steroid Delta5-->4-isomerase (3beta-HSD/isomerase) were purified as a single protein from human term placenta. The affinity alkylator, 5,10-secoestr-4-yne-3,10, 17-trione (secosteroid), was incubated with the purified enzyme (30/1 secosteroid/enzyme molar ratio) to produce an 80% loss of initial isomerase activity over 90 min in a time-dependent, irreversible manner. The secosteroid inactivated 3beta-HSD by only 20% during the same 90 min. Incubations containing the isomerase substrate steroid, 5-androstene-3,17-dione, completely protected the isomerase activity from inactivation by the secosteroid and did not slow the inactivation of 3beta-HSD. The enzyme containing covalently bound steroid was separated from unreacted secosteroid by reversed phase HPLC. Ketones on the protein-bound secosteroid were radiolabeled by reduction with sodium boro[3H]hydride (specific radioactivity 50 microCi/micromol for the transferred tritium). After removal of the unreacted sodium boro[3H]hydride, the affinity-radiolabeled enzyme was digested with trypsin-TPCK, and the peptides were isolated by reversed phase HPLC. The radiolabeled peptide fractions were sequenced. The secosteroid alkylated three tryptic peptides: 251GQFYYISDDTPHQSYDNLNYTLSK274, tritiated His262; 176NGGTLYTCALR186, tritiated Cys183; and 353TVEWVGSLVDR363, tritiated Trp356. Coincubation with the isomerase substrate blocked the labeling of these three peptides and shifted the alkylation by secosteroid to a single tryptic peptide (135EIIQNGHEEEPLENTWPAPYPHSK159, tritiated His142). Using substrate protection to validate specificity, the affinity labeling secosteroid has identified peptides in the enzyme that are associated with isomerase activity.

Physiological 3 beta-hydroxy-5-ene steroid substrates bind to 3 beta-hydroxysteroid dehydrogenase without the prior binding of cofactor.

3 beta-Hydroxy-delta 5-steroid dehydrogenase (3 beta-HSD)/steroid delta 5-4-isomerase catalyses the conversion of 3 beta-hydroxy-5-ene steroids (e.g. pregnenolone) to 3-oxo-4-ene-steroids (progesterone) in human placenta. Isotope exchange at equilibrium using NAD+/NADH and the 5 alpha-reduced steroids, 5 alpha-androstane-3 beta, 17 beta-diol and 5 alpha-androstan-17 beta-ol-3-one, determined a cofactor-first order of binding for these 3 beta-HSD substrates [1]. Exchange at equilibrium cannot be performed with 3 beta-hydroxy-5-ene steroids because 3 beta-HSD is not reversible with the 5-ene substrates. To compare their cofactor requirements for binding, 3 beta-hydroxy-5-ene and 3 beta-hydroxy-5 alpha-reduced steroids were tested as protectors against the inactivation of purified human placental 3 beta-HSD by 2 alpha-bromoacetoxyprogesterone (2 alpha-BAP) in the presence or absence of cofactor. In incubations without cofactor, pregnenolone or dehydroepiandrosterone dramatically slowed (protected) the rate of 3 beta-HSD inactivation by 2 alpha-BAP, an affinity alkylator that binds specifically at the 3 beta-HSD substrate site. In contrast, 5 alpha-androstan-3 alpha-ol-17-one, 5 alpha-androstane-3 beta, 17 beta-diol, or 11 alpha-acetoxy-5 alpha-pregnan-3,20-dione protected 3 beta-HSD from inactivation by 2 alpha-BAP only in the presence of NADH (0.3 microM) or NAD+ (10 microM). At these low concentrations, neither NADH nor NAD+ slowed the inactivation of 3 beta-HSD by 2 alpha-BAP in the absence of protector-steroid. Further, the 3-oxo-5 alpha-reduced alkylator, 11 alpha-bromoacetoxy-5 alpha-pregnan-3,20-dione (11 alpha-BA-5 alpha-P), did not inactivate 3 beta-HSD in a specific manner. After pre-incubation with NAD+ (10 microM), 11 alpha-BA-5 alpha-P inactivated 3 beta-HSD rapidly and specifically (t1/2 = 3.7 min). 11 alpha-Bromoacetoxyprogesterone inactivated 3 beta-HSD at the same rate (t1/2 = 5.0 min) in the presence or absence of NAD+. These affinity labelling studies confirm the cofactor-first binding order for 3 beta-hydroxy-5 alpha-reduced steroids, and conclusively show that the more important, physiological 3 beta-hydroxy-5-ene substrates bind to 3 beta-HSD without a cofactor requirement.

Endometrial thickness is a valid monitoring parameter in cycles of ovulation induction with menotropins alone.

OBJECTIVE: To evaluate the ability of an ultrasound (US)-measured periovulatory endometrial thickness to predict conception in hMG-stimulated cycles. DESIGN: Retrospective. SETTING: A university-based tertiary practice. PATIENTS: One hundred twelve patients undergoing 292 cycles of ovulation induction with hMG alone. MAIN OUTCOME MEASURES: A periovulatory transvaginal US measurement of endometrial thickness was obtained during cycles of ovulation induction with hMG alone. Clinical pregnancy was defined by fetal cardiac activity. Sensitivity and false-positive rates for multiple discriminatory values of endometrial thickness were calculated and a relative operating characteristic (ROC) curve was constructed to evaluate the performance of this test as a predictor of pregnancy. RESULTS: Thirty-eight of 292 cycles resulted in pregnancy. Conception and nonconception cycles showed similar demographics, diagnoses, peak E2, maximum number of follicles, midluteal P, and mean endometrial thickness. Ovulatory dysfunction was a more frequent diagnosis in the conception group. Relative operating characteristic analysis for endometrial thickness as a predictor of pregnancy yielded an area under the curve of 0.623 +/- 0.049 (mean +/- SD). CONCLUSION: Endometrial thickness is a valid screening test for conception outcome in cycles stimulated with hMG. A periovulatory endometrial thickness > or = 10 mm defined 91% of conception cycles. No pregnancy occurred when the endometrium measured < 7 mm.

An NADH-induced conformational change that mediates the sequential 3 beta-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and steroid delta-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 microM, Vmax = 0.6 mumol/min/mg), the affinity alkylating nucleotide, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (Kact = 338 microM, Vmax = 2.1 mumol/min/mg) prior to inactivating the enzyme. The inactivation kinetics for isomerase fit the Kitz and Wilson model for time-dependent, irreversible inhibition by 8-BDB-TADP (KI = 314 microM, first order maximal rate constant kobs = 7.8 x 10(-3) s-1). NADH (50 microM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 microM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 microM (t1/2 = 8.4 min) to 500 microM (t1/2 = 2.4 min). In sharp contrast, the 3 beta-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 microM (t1/2 = 4.8 min) to 500 microM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3 beta-HSD inactivation is a consequence of the activation of isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme conformation. Biophysical support for an NADH-induced conformational change was obtained using stopped-flow fluorescence spectroscopy. The binding of NADH (10 microM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 x 10(-3) s-1, t1/2 = 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3 beta-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.

Controlled ovarian hyperstimulation regimens in assisted reproductive technologies.

The goal of assisted reproductive technologies (ART) is to influence the recruitment of multiple, mature ovarian follicles. Several methods, including spontaneous cycle ART, clomiphene-based ART regimens, and gonadotropin regimens with and without adjuncts, are used. The controversies surrounding these techniques and their relative advantages and drawbacks are reviewed.

Over-expression of human type I (placental) 3 beta-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus.

Human type I placental 3 beta-hydroxy-5-ene-steroid dehydrogenase/steroid 5-->4-ene-isomerase (3 beta-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3 beta-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3 beta-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3 beta-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3 beta-HSD substrate, 5 alpha-androstan-3 beta- ol-17-one, in the Sf-9 cell homogenate (Km = 17.9 microM) and placental microsomes (Km = 16.7 microM). The 3 beta-HSD activity (Vmax = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (Vmax = 9.1 nmol/min/mg). The Km values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (Km = 14.7 microM) and placental microsomes (Km = 14.4 microM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (Vmax = 25.7 nmol/min/mg) relative to placenta (Vmax = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3 beta-HSD/isomerase in human placenta.

Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[14C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkylated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. RESULTS: According to the sequence analyses, NADH shifted radioalkylation by 2 alpha-[14C]BAP away from the Arg-250 peptide (251GQFYYISDDTPHQSYDNLNYTLSK274) and toward the Lys-135 tryptic peptide (136EIIQNGHEEEPLENTWPAPYPHSK159). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His262 in the Arg-250 peptide by 8.2-fold. His142 in the Lys-135 peptide was radiolabeled by 2 alpha-[14C]BAP only in the presence of NADH. CONCLUSIONS: We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[14C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.

An unusual active hexose transport system in human and mouse preimplantation embryos.

In a metabolic study of human and mouse preimplantation embryos (preembryos), we measured glucose uptake and phosphorylation with nonradioactive 2-deoxyglucose (DG) as tracer. Initial experiments indicated an active hexose transport capacity, a property thought to be restricted in mammals to intestinal villi and kidney tubules [Baly, D. L. & Horuk, R. (1988) Biochim. Biophys. Acta 947, 571-590]. Significant findings are as follows: (i) During a 60-min incubation with a low level of DG, mouse blastocyst DG rose to levels up to 30 times that of the medium. (The intestinal active system does not transport DG [Crane, R. K. (1960) Physiol. Rev. 40, 789-825].) (ii) Active preembryo transport was not blocked (as it would have been in the intestine) by phlorizin [Alvarado, F. & Crane, R. K. (1962) Biochem. Biophys. Acta 56, 170-172 and Sacktor, B. (1989) Kidney Int. 36, 342-350] or by replacement of Na+ with choline+ or K+ [Crane (1960) and Sacktor (1989)]. (iii) Transport of DG was blocked by cytochalasin B (which is not true for the intestinal transporter). We conclude that a distinct active hexose transporter and at least one facilitated transporter are present in preembryos, perhaps appearing in tandem on different membranes during formation of the increasingly complex preembryo structure.

Affinity radiolabeling identifies peptides and amino acids associated with substrate binding in human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase.

Purified human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase (3 beta-HSD) was affinity radiolabeled by 2 alpha-bromo[2'-14C]acetoxyprogesterone (2 alpha-BAP) in the presence or absence of 3 beta-HSD substrate, pregnenolone. The substrate steroid substantially protects 3 beta-HSD activity from inactivation by 2 alpha-BAP. Tryptic peptides of unprotected and substrate-protected radioalkylated enzyme were purified by high pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. According to the percent total radioactivity associated with each of four radiolabeled peaks separated by high pressure liquid chromatography, two peptides were protected by substrate from affinity radioalkylation by 2 alpha-BAP. The first, 251GQFYYISDDTPHQSYDNLNYTLSK274, was produced by tryptic cleavage at Arg-250 and Lys-274 (the Arg-250 peptide) and contained radiolabeled His262. The second, 176NGGTLYTCALR186, was produced by tryptic cleavage at Lys-175 and Arg-186 (the Lys-175 peptide) and contained radiolabeled Cys183. Based on amino acid analysis to quantitate radioactivity incorporated per nmol of peptide, substrate steroid decreased the radiolabeling of His262 in the Arg-250 peptide by 3.6-fold and decreased the radiolabeling of Cys183 in the Lys-175 peptide by 3.7-fold. Three minor radiolabeled peptides (the NH2-terminal, Arg-71, and Arg-196 tryptic peptides) were also identified in the primary structure, but pregnenolone did not diminish their affinity radioalkylation. These observations indicate that the Arg-250 and Lys-175 peptides are involved in substrate binding and suggest that His262 and Cys183 are in close proximity in the three-dimensional structure of the enzyme.

Enzyme activities and maturation in unstimulated and exogenous gonadotropin-stimulated human oocytes.

With the advent of new techniques of human in vitro fertilization (IVF), identifying parameters of oocyte quality to allow selection of those most likely to fertilize becomes crucial. Morphology of oocytes, which correlates positively with biological performance, is the currently utilized classification criterion. However, biological links between form and function are tenuous, and underlying mechanisms remain elusive. We investigated whether biochemical activation is quantitatively associated with the stages of maturation in ova obtained from patients undergoing gynecologic surgery during unstimulated cycles and women undergoing IVF after exogenous gonadotropin stimulation. Changes in selected enzymes from protein, lipid, and carbohydrate metabolism (hexokinase, phosphoglucomutase, glycogen synthetase, uridine diphosphoglucose pyrophosphorylase, glucose-6-phosphate dehydrogenase, cytosolic thiolase, beta-hydroxyacyl-CoA dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) were determined simultaneously, in individual oocytes, utilizing a highly sensitive biochemical methodology. Several enzyme activities paralleled maturation grade and were higher in stimulated oocytes after correction for grade. These biochemical findings quantify metabolic and functional changes that increase as ova mature, possibly contributing to their reproductive performance.

Affinity labeling identifies histidine at the active site of human placental 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase.

OBJECTIVE: Our aim was to determine if the multifunction enzyme, 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase has one or more active sites to effect dehydrogenase and isomerase activities. STUDY DESIGN: This steroid, which we have purified to homogeneity from human placental microsomes, was inactivated by the affinity labeling steroid, 2 alpha-bromo[2'-14C]acetoxyprogesterone. The amino acids that were radioalkylated in the absence and presence of the dehydrogenase substrate pregnenolone were identified. RESULTS: Pregnenolone completely abolished the inactivation of dehydrogenase. Histidine was localized in the active site of 3 beta-hydroxysteroid dehydrogenase because the radiolabel disappeared from enzyme inactivated in the presence of pregnenolone. Cysteine, a major radiolabeled product (80%) in the absence of pregnenolone, was decreased twofold in incubations that contained pregnenolone. Neither pregnenolone nor the isomerase substrate 5-androstene-3,17-dione protected isomerase from inactivation by the affinity alkylator. CONCLUSION: This observation contradicts coexisting, separate binding sites, one for each activity. Rather, a conformation shift around one binding region prompted by products of the dehydrogenase reaction may create the isomerase activity.

Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.

3 beta-Hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3 beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.

Analysis of coenzyme binding by human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase using 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an affinity labeling cofactor analog.

3 beta-Hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase copurify as a single, homogeneous protein from human placental microsomes. Affinity alkylation with 2 alpha-bromoacetoxyprogesterone suggests that the dehydrogenase and isomerase substrate steroids bind at different sites on the same protein. However, the coenzyme, NADH, completely abolishes the alkylation of both enzyme activities by the progestin analog [Thomas J .L., Myers R. P., Rosik L. O. and Strickler R. C., J. Steroid Biochem. 36 (1990) 117-123]. Unlike bacterial 3-keto-5-ene-steroid isomerase, the human isomerase reaction is stimulated by diphosphopyridine nucleotides (NADH, NAD+). The affinity labeling nucleotide analog, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSA), inactivates the dehydrogenase and isomerase activities at similar rates in an irreversible manner which follows first order kinetics with respect to both time and alkylator concentration (0.2-0.6 mM). FSA is a cofactor site-directed reagent that binds with similar affinity as a competitive inhibitor of NAD+ reduction by dehydrogenase (Ki = 162 microM) or as a stimulator of isomerase (Km = 153 microM). Parallel plots derived from Kitz and Wilson analysis indicate that FSA inactivates the two enzyme activities with equal alkylation efficiency (k3/Ki = 1/slope = 0.51/mol-s for both). The 3 beta-hydroxysteroid substrate, pregnenolone, protects isomerase as well as dehydrogenase from inactivation by FSA. These observations are evidence for a single cofactor binding region which services both enzyme activities.

Therapeutic donor insemination: a prospective randomized study of scheduling methods.

OBJECTIVE: To compare basal body temperature (BBT) graphs and urinary luteinizing hormone (LH) monitoring in scheduling therapeutic donor insemination. DESIGN: Participants were prospectively randomized to the BBT or LH groups. SETTING: Participants were private patients of the Reproductive Endocrine Division at Washington University School of Medicine. PATIENTS: Inclusion criteria were designed to assure an isolated male factor. Seventy-four of 113 patients completed the study; 18 had ongoing treatment at the end of the study. INTERVENTIONS: Basal body temperature graphs were physician interpreted and appointments prospectively chosen. Luteinizing hormone patients monitored daily urine samples and scheduled an appointment the day after the detected surge. MAIN OUTCOME MEASURES: Fecundity rates, cumulative pregnancy rates, and cost per pregnancy were all prospectively evaluated. RESULTS: Life table analysis yielded a 6-month cumulative probability of pregnancy of 36.3% in the LH group and 65.1% in the BBT group (P less than 0.025). The total cost per pregnancy was lower in the BBT group (+6,212 versus +3,997; P less than 0.001). CONCLUSIONS: This randomized prospective study demonstrates significant therapeutic and economic advantages when therapeutic donor insemination is prospectively scheduled by BBT graphs.

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein.

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland.

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.