Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol

14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.     

Isolation of novel microbial 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Purification, characterization, and analytical applications of a 17 beta-hydroxysteroid dehydrogenase from an Alcaligenes sp

By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta-hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3 alpha-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 17 beta-hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM). It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17 beta-hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions. Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function. 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates. The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated.     

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.     

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)     

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.     

Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.

Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.     

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17 alpha-estradiol 17-(bromo[2-14C]acetate) (10 microM) or 17 beta-estradiol 17-(bromo[2-14C]acetate) (10 microM). The steroid bromoacetates competitively inhibit the enzyme (against 17 beta-estradiol) with Ki values of 90 microM (17 alpha bromoacetate) and 134 microM (17 beta bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t1/2 = 110 min (17 alpha bromoacetate) and t1/2 = 220 min (17 beta bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N pi-(carboxy[14C]methyl)histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N pi-(carboxy[14C]methyl)histidine. Both the 17 alpha bromoacetate and also the 17 beta bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(pi-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17 alpha bromoacetate and 17 beta bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of a steroidal A ring aromatic sulfonic acid as an inhibitor of steroid 5 alpha-reductase

The synthesis of 17 beta-(N,N-diisopropylcarbamoyl)estra-1,3,5(10)-triene-3-sulfonic acid (3) has been accomplished. Sulfonate 3 was designed as a novel inhibitor of human steroid 5 alpha-reductase based on considerations of enzyme mechanisms, and exhibits an inhibition constant in the low nanomolar range.     

Reactivation of human placental 17β, 20α-hydroxysteroid dehydrogenase: Affirmation of affinity labeling principles

Human placental 17β, 20α-hydroxysteroid dehydrogenase was completely inactivated by the affinity alkylator, 3-bromoacetoxy-1,3,5(10)-estratrien-17-one (estrone 3-bromoacetate). The inactivated enzyme was then reactivated to 100% of the enzyme activity by base-catalyzed hydrolysis of the steroidalester-enzyme conjugate. After the reactivated enzyme was repurified by dialysis, re-inactivation studies were performed on it. The reactivated enzyme could not be re-inactivated by the original alkylator, estrone 3-bromoacetate. However, 16α-bromoacetoxyestradiol-17β 3-methyl ether caused a loss of reactivated enzyme activity at a rate comparable to that for the native enzyme. These observations demonstrate that a specific amino acid modification within the enzyme active site was produced by estrone 3-bromoacetate alkylation and suggest that the conformation of the active center was essentially unaltered. Thus, these successful reactivation studies of 17β, 20α-hydroxysteroid dehydrogenase affirm the specificity of affinity labeling. This methodology also offers a new tool to investigate the steroid binding regions of macromolecular proteins.     

Hydroxysteroid dehydrogenases of Pseudomonas testosteroni. Separation of a 17 beta-hydroxysteroid dehydrogenase from the 3(17) beta-hydroxysteroid dehydrogenase and comparison of the two enzymes.

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

17 Beta-hydroxysteroid dehydrogenase in human breast cancer: analysis of kinetic and clinical parameters

In this study, we assessed the rate of estradiol degradation via the 17 beta-hydroxysteroid dehydrogenase (HSD) enzyme in breast tumors from postmenopausal women. We initially studied the effects of time, level of enzyme activity, amount of tissue assayed, and substrate concentration on the linearity of conversion of estradiol to estrone in breast tumor homogenates. The reaction was demonstrated to be linear when less than 15% conversion of estradiol to estrone occurred over 30 min with homogenates produced from 2.5 mg of tissue. Detailed kinetic experiments demonstrated the presence of two classes of enzyme activity, one with high affinity and the other with low affinity. In 83% of the tumors examined, the high affinity form was present and had a median Km of 0.62 microM and Vmax of 82 nmol/g protein/h. In 29 tumors, HSD activity could be precisely quantified and correlated with clinical parameters. No statistically significant correlation of enzyme activity with estrogen receptor (r2 = 0.06) or progesterone receptor (r2 = 0.006) or with patient age could be detected (r2 = 0.001). In 12 additional tumors, activity exceeded 15% conversion of estradiol to estrone at 30 min and precise quantitation was not possible. The average content of progesterone receptor was similar for these 12 tumors as for the 19 with lower HSD activity. However, estrogen receptor content and patient age were lower in the group with high HSD activity. The finding of a high affinity form of HSD in this study provides support for the biological importance of this enzyme in breast cancer tissues.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.