Affinity labeling of cofactor-binding region of human placental estradiol 17 beta-dehydrogenase by periodate-oxidized NADP+ (o-NADP+)

Periodate-oxidized NADP+ (o-NADP+), an analogue of the cofactors, is a reversible inhibitor of estradiol 17 beta-dehydrogenase in human placenta. Mode of the inhibition by o-NADP+ appeared to be competitive type (Ki = 0.84 microM) against NAD+ and non-competitive type (Ki = 1.13 microM) against estradiol, respectively. Treatment of the estradiol 17 beta-dehydrogenase with o-NADP+ resulted in time-dependent loss of the enzyme activity. The inactivation exhibited pseudo-first order kinetics (t1/2 = 15 min) and was protected by NAD+ and NADP+. On the other hand, periodate-oxidized ATP inactivated slightly the estradiol 17 beta-dehydrogenase. These results indicate that the residue(s) of lysines is located near the cofactor-binding region of estradiol 17 beta-dehydrogenase of human placenta.     

Affinity labeling of the cofactor-binding site of estradiol 17 beta-dehydrogenase of human placenta by 5'-p-fluorosulfonylbenzoyl adenosine

An analogue of adenosine nucleotide, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSB-Ado), appears to interact irreversibly with the cofactor-binding site of estradiol 17 beta-dehydrogenase of human placenta. This conclusion is based on the following observations: (1) The estradiol 17 beta-dehydrogenase is inhibited by 5'-FSB-Ado. When NAD+ is the variable component in the presence of saturated amount of steroid, the type of the inhibition is competitive in nature. When the steroid is the variable component, mode of the inhibition becomes non-competitive. The results suggest reversible binding of 5'-FSB-Ado to the cofactor-binding site of the dehydrogenase. (2) 5'-FSB-Ado inactivates irreversibly the estradiol 17 beta-dehydrogenase in time- and concentration-dependent manners, following pseudo-first-order kinetics. But, no inactivation is observed in the presence of p-fluorosulfonylbenzoic acid, suggesting that adenosine moiety of 5'-FSB-Ado is essential for the affinity labeling of estradiol 17 beta-dehydrogenase. (3) NADP+ protects completely estradiol 17 beta-dehydrogenase from the inactivation of 5'-FSB-Ado, whereas NAD(H) is partially protective against the inactivation, suggesting that phosphate moiety at 2'-position of NADP+ disturbs the covalent binding of 5'-FSB-Ado at or near the cofactor-binding site of the enzyme. (4) 2',5'-ADP shows the significant protection against the inactivation by 5'-FSB-Ado, but less effect is observed in the presence of nicotinamide mononucleotides. These results suggest that 5'-FSB-Ado is an affinity ligand for binding-site of adenosine nucleotide moiety of the cofactor.     

Synthesis of 16alpha-bromoacetoxyestradiol 3-methyl ether and study of the steroid binding site of human placental estradiol 17beta-dehydrogenase.

Homogeneous estradiol 17beta-dehydrogenase (EC 1.1.1.62) was prepared from human placenta by affinity chromatography and the steroid binding site was studied with affinity-labeling techniques. 16alpha-Bromoacetoxyestradiol 3-methyl ether and the tritated compound were synthesized by condensation of estriol 3-methyl ether with bromoacetic acid or [2-3H]bromoacetic acid in the presence of dicyclohexylcarbodiimide. 16alpha-Bromoacetoxyestradiol 3-methyl ether is stable in 0.01 M phosphate buffer at pH 7.0, 25 degrees, for at least 24 hours. It alkylates cysteine, histidine, methionine, lysine, and tryptophan under physiological conditions. The steroid is a substrate of estradiol 17beta-dehydrogenase, thus it must bind at the steroid binding site. The inactivation of estradiol 17beta-dehydrogenase by 150-fold molar concentrations of 16alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo-first order kinetics with a half-time of 1.5 hours. Estradiol-17beta, NADH, and NADPH slow the rate of inactivation. 2-Mercaptoethanol in molar concentrations 50-fold that of 16alpha-bromoacetoxyestradiol 3-methyl ether stops the inactivation, but does not reverse it. 16alpha-Bromoacetoxyestradiol 3-methyl ether alkylates both NADH and NADPH; the presence of small amounts of enzyme markedly increases the rate of this alkylation. When the enzyme is inactivated with 16alpha-[2-3H]bromoacetoxyestradiol 3-methyl ether, amino acid analysis of acid hydrolysates reveals 3-carboxymethylhistidine and 1,3-dicarboxymethylhistidine. Comparison of 28 and 51% inactivated samples indicates that, as inactivation proceeds, the total amount of 3-carboxymethylhistidine decreases, while 1,3-dicarboxymethylhistidine increases, suggesting that the former is converted to the latter by a second alkylation step. When the enzyme is inactivated in the presence of a large excess of NADPH, only 1,3-dicarboxymethylhistidine is found. From the present study it is concluded that estradiol 17beta-dehydrogenase has a histidyl residue present in the catalytic region of the active site as does the previously studied 20beta-hydroxysteroid dehydrogenase.     

Functional site of human placental estradiol 17 beta-dehydrogenase involves tyrosine residues, as evidenced by reaction to p-nitrobenzenesulfonyl fluoride

Native estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from human placenta was inactivated in time dependent manner by p-nitrobenzenesulfonyl fluoride (NBSF), which is a reagent for chemical modification of tyrosine. The sulfhydryl-blocked enzyme by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) was also reacted with NBSF more slowly in pseudo-first-order kinetics. After the sequential treatments with DTNB, NBSF and dithiothreitol (DTT), the enzyme in which tyrosine residues alone were modified was isolated, and its activity was decreased. These results suggest that tyrosyl residues of the estradiol 17 beta-dehydrogenase from human placenta are located at or near its catalytic site, and play a functional role in the enzyme reaction.     

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

Chemical modification of lysine residues at active-site of human placental estradiol 17 beta-dehydrogenase

Estradiol 17 beta-dehydrogenase (EC 1.1.1.62.) activity was decreased by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent for modification of epsilon-amino moiety of lysine residues in a protein. The inactivation exhibited pseudo-first-order kinetics, and was protected by oxidyzed cofactors. Stoichiometric studies showed that the complete inactivation was caused by modification of one lysine residue per molecule of the enzyme. Differential modification with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), TNBS and dithiothreitol (DTT) indicated that the residues of lysine and cysteine were located at the active-site and played an essential role in the catalytic function of the estradiol 17 beta-dehydrogenase.     

Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region

The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.     

Guinea-pig liver testosterone 17 beta-dehydrogenase (NADP+) and aldehyde reductase exhibit benzene dihydrodiol dehydrogenase activity.

We have kinetically and immunologically demonstrated that testosterone 17 beta-dehydrogenase (NADP+) isoenzymes (EC 1.1.1.64) and aldehyde reductase (EC 1.1.1.2) from guinea-pig liver catalyse the oxidation of benzene dihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene) to catechol. One isoenzyme of testosterone 17 beta-dehydrogenase, which has specificity for 5 beta-androstanes, oxidized benzene dihydrodiol at a 3-fold higher rate than 5 beta-dihydrotestosterone, and showed a more than 4-fold higher affinity for benzene dihydrodiol and Vmax. value than did another isoenzyme, which exhibits specificity for 5 alpha-androstanes, and aldehyde reductase. Immunoprecipitation of guinea-pig liver cytosol with antisera against the testosterone 17 beta-dehydrogenase isoenzymes and aldehyde reductase indicated that most of the benzene dihydrodiol dehydrogenase activity in the tissue is due to testosterone 17 beta-dehydrogenase.     

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.     

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)     

Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens

Antiinflammatory agents and estrogens have been tested as inhibitors of two isozymes of guinea pig liver testosterone 17 beta-dehydrogenase (NADP) 1.1.1.64) and rat liver 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50). Antiinflammatory steroids and estradiols were highly inhibitory to 3 alpha-hydroxysteroid dehydrogenase and one isozyme of testosterone 17 beta-dehydrogenase, respectively, but nonsteroidal antiinflammatory agents and nonsteroidal estrogens such as hexestrol, dienstrol, diethylstilbestrol and zearalenone showed potent inhibitions on all the enzymes. Although the inhibitory potency of indomethacin for one isozymes of testosterone 17 beta-dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase decreased with changing pH from 9.7 to 7.0, that of the nonsteroidal estrogens for all the enzymes was little affected by pH. No additive effect in double inhibitor experiments with indomethacin and the nonsteroidal estrogens was observed, and the compounds were all competitive inhibitors with respect to steroidal substrate. The results suggest that there is a very similar region in substrate binding sites of the enzymes.     

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.     

Alkylation of estradiol 17 beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide phosphate.

3-Chloroacetylpyridine--adenine dinucleotide phosphate is both active as a hydride acceptor and inactivates estradiol 17 beta-dehydrogenase. This coenzyme analogue behaves like an affinity label. The inactivation kinetics are discussed in relation to those observed with 3-chloroacetylpyridine--adenine dinucleotide. The pH dependence of the rate of inactivation, in combination with determination of the number of reactive cysteine residues, pointed to the alkylation of one cysteine residue/subunit. The stoichiometry was one molecule of dinucleotide per subunit and no cooperativity was detected. When 14C-labeled dinucleotide was used, the 14C label was found mainly in one peptide, accounting for 90% of the incorporated radioactivity, whereas in previous work it had been shown that 3-chloroacetylpyridine--adenine dinucleotide is an affinity reagent which labels three peptides.     

Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]

Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase.     

Cloning and sequencing of cDNA encoding human sepiapterin reductase--an enzyme involved in tetrahydrobiopterin biosynthesis

A full-length cDNA clone for sepiapterin reductase, an enzyme involved in tetrahydrobiopterin biosynthesis, was isolated from a human liver cDNA library by plaque hybridization. The nucleotide sequence of hSPR 8-25, which contained an entire coding region of the enzyme, was determined. The clone encoded a protein of 261 amino acids with a calculated molecular mass of 28,047 daltons. The predicted amino acid sequence of human sepiapterin reductase showed a 74% identity with the rat enzyme. We further found a striking homology between human SPR and carbonyl reductase, estradiol 17 beta-dehydrogenase, and 3 beta-hydroxy-5-ene steroid dehydrogenase, especially in their N-terminal region.     

Purification and properties of a new testosterone 17beta-dehydrogenase (NADP+) from guinea-pig liver.

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.     

Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide.

3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.     

Hydrogen bonding in steroidogenesis: studies on new heterocyclic analogs of estrone that inhibit human estradiol 17 beta-dehydrogenase

New heterocyclic analogs of estrone are reported that inhibit estradiol 17 beta-dehydrogenase (E2-17 beta DH) from human placenta. The inhibitors are efficiently synthesized in two steps from estrone (or its 3-O-methyl ether), giving fully characterized analogs with pyrazole or isoxazole fused to the 16,17-position on the D ring. Dixon plots of enzyme kinetic data show the heterocyclic steroids are competitive inhibitors of E2-17 beta DH. Correlating molecular structures of the inhibitors with their Ki-values yields a pattern suggesting intermolecular hydrogen bonding stabilizes the [(pyrazole)inhibitor-E2-17 beta DH] complexes. A free energy difference of 2.74 Kcal/mol calculated from Ki-value differences between hydrogen bonded (4.08 microM) and non-bonded (425 microM) [inhibitor-E2-17 beta DH] complexes is in the range for intermolecular hydrogen bonding. We conclude that specific intermolecular hydrogen bonds stabilize [hydroxysteroid-enzyme] complexes, thereby making important contributions to the affinity between hydroxysteroids and steroid-specific enzymes of steroidogenesis.     

A 17-kDa protein induced in rat myometrium by hormonal treatment

A 17-kDa protein mainly recovered in the myometrium fraction sedimenting at 10,000 g has been induced by estradiol treatment in spayed rats. Tamoxifen completely inhibited the effect of estradiol, and progesterone decreased it by 50%. The protein was scarce in unspayed animals and the level after estradiol treatment remained much lower than in spayed rats. Oral doses of the potent antiprogesterone RU 38486 increased the level of the 17-kDa protein in unspayed animals. It was not present in immature or gestating rats or in the endometrium or myometrial cell cytosol of any of the rats. Following electrophoretic purification, it bound calmodulin, but was not phosphorylated by cAMP protein kinases.