PubMed Health⌕ Search

Biomedical subjects

R C Strickler

Publications and source records attributed to R C Strickler.

At least 19 recordsLinked to original sources
PubMed1992

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

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.

Body Temperature↗
PubMed1990

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-Hydroxysteroid Dehydrogenases↗
PubMed1990

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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes.

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

Ovarian stimulation with pure follicle-stimulating hormone/human menopausal gonadotropin and improved laparoscopic aspiration needles influence the success of an in vitro fertilization program.

The effect of a combined pure follicle-stimulating hormone/human menopausal gonadotropin (pFSH/hMG) ovarian stimulation regimen and modified, sharpened laparoscopic follicular aspiration needles on the number of oocytes retrieved and the oocyte/follicle ratio in 43 consecutive cycles of in vitro fertilization (IVF) were retrospectively compared with 99 consecutive preceeding cycles stimulated with hMG alone and captured with aspiration needles that had never been sharpened. A modified laparoscopic follicular aspiration needle is described. Purified FSH/hMG ovarian stimulation significantly improved the mean serum estradiol levels, number of preovulatory follicles, and, therefore, the total number of oocytes recovered per cycle. The mean ratios of oocytes recovered per preovulatory follicle documented on ultrasound, and per aspirated follicle, increased significantly using sharpened needles. Both modifications improved the success rate of our IVF program.

Female↗
PubMed1989

Full length cDNA structure and deduced amino acid sequence of human 3 beta-hydroxy-5-ene steroid dehydrogenase.

Polyclonal antibodies raised against 3 beta-hydroxysteroid dehydrogenase isolated from human placenta were used to screen a lambda gt11 expression cDNA library from the same tissue. The protein deduced from cDNA sequences contains 372 amino acids with a calculated mol wt of 42,216. Since 3 beta-hydroxysteroid dehydrogenase is the enzyme catalyzing the formation of all classes of hormonal steroids, the availability of the cDNA encoding this enzyme opens new possibilities for a detailed investigation of the factors regulating the expression and activity of this crucial enzyme in adrenal, gonadal as well as peripheral tissues.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids.

In human pregnancy, placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric Mr = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (Mr = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37 degrees C for the oxidation of pregnenolone (Km = 1.9 microM, Vmax = 32.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.8 microM, Vmax = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.7 microM, Vmax = 618.3 nmol/min/mg) and 5-androstene-3,17-dione (Km = 23.7 microM, Vmax = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Laser vaporization of endometriosis in an infertile population: the role of complicating infertility factors.

One hundred twenty-two infertility patients with endometriosis were evaluated and treated using laparoscopy and laser vaporization to provide immediate elimination of all intraperitoneal disease. Ninety-five couples (77.8%) had one or more infertility factors (other than endometriosis) contributing to their infertility. Cervical factors, male factors, and luteal defects were associated with significantly decreased pregnancy rates, despite the use of laser vaporization. The number of contributing factors seemed to be unrelated to the likelihood of success in achieving pregnancy. The authors emphasize the need for total evaluation of other infertility factors in these patients. Such factors should be corrected prior to the use of laser laparoscopy, if possible, and dealt with on a continuing basis following use of this technique.

Endometriosis↗
PubMed1988

Contrast in levels of metabolic enzymes in human and mouse ova.

A methodology is described for analyzing single human ova for 8 or 9 different metabolic enzymes, or 4 or 5 enzymes plus as many metabolites. This overcomes an obstacle to the study of human ovum metabolism: the severe limitation of usable material. Results obtained with this methodology, applied to discarded specimens from an in vitro fertilization program, indicate that in spite of imperfections these ova can provide a valid picture of the metabolic characteristics of normal human ova. Data are presented for 17 enzymes from 8 metabolic pathways in human and mouse ova. Relative to size, 10 of the enzymes were substantially higher in human than mouse ova. Most dramatically so were 2 enzymes of fatty acid metabolism (10-fold and 15-fold), hexokinase (9-fold), and aspartate aminotransferase (19-fold). This suggests that major species differences in metabolism are present. The validity of the human data, in spite of restriction to discarded material, is supported by (1) consistency of results among most of the ova, 2] concordance between average levels with those of rare specimens that were discarded because sperm were not available, and (3) the presence of adenosine triphosphate (ATP) concentrations similar to those of normal mouse ova. Surprisingly, both human and mouse ova contain phosphocreatine at levels nearly equal of those of ATP.

Acetyl-CoA C-Acetyltransferase↗
PubMed1988

Augmentation of the response of mouse uterine epithelial cells to estradiol by uterine stroma.

The effect of estrogen on the in vitro growth of mouse uterine epithelial cells was assessed. Epithelial cells from the immature mouse uterus were successfully cultivated in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F-12 supplemented with insulin (5 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (10(-7) M), BSA (2 mg/ml), and fetuin (1 mg/ml). Addition of 17 beta-estradiol in the range of 1-100 nM did not significantly change the total DNA content of the epithelial cells. A binding component of [3H]estradiol by cultured uterine cells was shown to be specific, saturable, and of high affinity. Kd values for specific binding by epithelial and stromal cells were 1.0-1.7 x 10(-10) M. Maximal specific binding was 0.74 and 2.3 fmol/micrograms DNA for epithelial and stromal cells, respectively. Treatment of epithelial and stromal cells for 4 days with 10 nM estradiol led to a 2- to 6-fold increase in progesterone receptor concentration. Treatment of epithelial and stromal cells in mixed culture for 4 days with 10 nM estradiol resulted in a significant increase in total DNA. That epithelial-stromal contact was critical for estradiol stimulation was shown by the fact that if the cell types were separated into two compartments which still allowed free media mixing, total DNA was not enhanced by estradiol. These observations are organized into a model for mitogenic action of estradiol that seems to reconcile observed disparities in the action of the hormone in vivo and in vitro.

Animals↗
PubMed1987

A modified digitonin-precipitation radioassay for 3 beta-hydroxy-delta 5-steroid dehydrogenase.

An improved digitonin-precipitation radioassay for 3 beta-hydroxy-delta 5-steroid dehydrogenase is presented. It is linear for enzyme protein concentrations up to 0.6 mg and for 37 degrees C incubation times up to 10 min. Sensitivity and reproducibility are improved nine- and sevenfold, respectively, over the method of Philpott and Peron. There is a good correlation with the Philpott and Peron assay (r = 0.958) and with a thin-layer chromatography method (r = 0.997). Beyond being faster, simpler, and more sensitive than other analyses, this radioassay uses one-tenth the quantity of [3H]pregnenolone substrate and is, therefore, a safer and less expensive procedure.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

The affinity alkylators, 11 alpha-bromoacetoxyprogesterone and estrone 3-bromoacetate, modify a common histidyl residue in the active site of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase.

Purified human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (native enzyme) was completely inactivated by the affinity alkylator, estrone 3-bromoacetate, in the presence of cofactor (NADPH). The inactivated enzyme was reactivated to 100% activity by base-catalyzed hydrolysis of the steroidal ester-enzyme conjugate and then repurified by dialysis. Control enzyme in mixtures which contained estrone in place of alkylator was treated the same as the reactivated enzyme. 11 alpha-Bromo[2'-14C]acetoxyprogesterone, an active site-directed affinity alkylator of the enzyme, produced 5.0-fold less radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine plus 1.4-fold more 1,3-bis(carboxymethyl)-histidine in the reactivated enzyme than in the control enzyme. The lesser amount of S-(carboxymethyl)cysteine and greater amount of 1,3-bis(carboxymethyl)histidine resulted from nonspecific interactions between the reactivated enzyme and the progestin radioalkylator. The nonradiolabeled 3-(carboxymethyl)histidine originally produced by estrone 3-bromoacetate in the enzyme active site hindered radioalkylation of this amino acid by 11 alpha-bromo[2'-14C]acetoxyprogesterone to yield 5-fold less radiolabeled 3-(carboxymethyl)histidine in the reactivated enzyme relative to control enzyme. Thus, the estrogen and progestin affinity alkylators modified a common histidyl residue in the active site. These studies are direct evidence that the estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities reside at a common locus on a single protein.

Alkylation↗
PubMed1986

3 beta-Hydroxysteroid dehydrogenase in human placental microsomes and mitochondria: co-solubilization of androstene and pregnene activities.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) was solubilized from human term-placental microsomes and mitochondria using the non-ionic detergent, polyoxyethylene 20 cetyl ether (BrijR-58). Electron photomicrographs showed microsomes and mitochondria well disrupted by the detergent. The pregnene (C-21) and androstene (C-19) activities co-solubilized over a range (0.04-0.44) of BrijR-58/protein (B/P) concentration ratios (w/w). Optimal solubilization of the C-19 and C-21 activities were 63.3 +/- 2.6% (mean +/- SEM) from mitochondria (B/P ratio 0.37) and 71.8 +/- 2.1% from microsomes (B/P ratio 0.34). Detergent treatment of microsomes and mitochondria--varying time (5-90 min, pH 7.4) or varying pH (6.0-7.8, 90 min)--yielded C-19 activities identical with C-21 activities. The C-21/C-19 specific activity ratios of 3 beta-HSD in particulate, solubilized and chromatographed preparations were 2.28 +/- 0.16 (mean +/- SEM) for mitochondria and 1.97 +/- 0.07 for microsomes. 3 beta-HSD molecular weight estimates were 208,000 (microsomes) and 220,000 (mitochondria). These studies argue that a single protein is responsible for both the C-19 and C-21 activities of 3 beta-HSD and that this protein is the same in microsomes and mitochondria.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Ovulation induction with low doses of clomiphene citrate.

Ovulation was induced with one-quarter tablet (12.5 mg) of clomiphene citrate given for five days to three oligoovulatory patients who had consistently formed functional ovarian cysts when given 50 and 25 mg of clomiphene citrate for five days. Two of the patients had polycystic ovary syndrome and the other had hypothalamic dysfunction associated with an eating disorder. On clomiphene citrate 12.5 mg for five days, one patient conceived, another produced inadequate cervical mucus but later conceived after in vitro fertilization and embryo transfer, and the third woman, who had concomitant fallopian tube disease, ovulated but has not conceived. Women oversensitive to clomiphene citrate can be effectively treated with very low doses of the drug. Perhaps other oligoovulatory patients may benefit from lower than currently recommended clomiphene citrate doses.

Adult↗
PubMed1985

Reactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether).

Estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, oxidoreductase activities copurified from the cytosol of human-term placenta as a homogeneous protein (native enzyme), were reactivated at equal rates to 100% activity following complete inactivation in the presence of cofactor (NADPH) with the affinity alkylator estrone 3-(bromoacetate). Reactivation was accomplished by base-catalyzed hydrolysis of steroidal ester-amino acid linkages in the enzyme active site. The rate of enzyme reactivation was pH dependent. In identical studies without NADPH, only 12% of the original enzyme activity was restored. Completely reactivated enzyme was repurified by dialysis. Enzyme in control mixtures (control enzyme) that contained estrone in place of alkylator was treated the same as the reactivated enzyme. Reactivated enzyme exhibited a 6.0-fold lower affinity for common substrates, a 1.8-fold lesser affinity for NAD+ and NADH, and the same affinity for NADP+ and NADPH compared to control enzyme. In incubations that included NADPH, the reactivated enzyme maintained full activity during a 20-h second exposure to estrone 3-(bromoacetate), but in identical incubations without NADPH, the reactivated enzyme was rapidly inactivated at the same rate as the control and native enzymes. The control and reactivated enzymes were inactivated at equal rates by 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether) in the presence or absence of cofactor (NADP+) and exhibited similar Kitz and Wilson inhibition constants for this affinity alkylator. Estrone 3-(bromo[2'-14C]acetate) incubated with native enzyme and NADPH produced radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)

17-Hydroxysteroid Dehydrogenases↗