Characterization of two uterine proteases and their actions on the estrogen receptor

We have characterized two previously undetected proteases from the calf uterine cytosol and measured their actions on the estrogen receptor. One is an exopeptidase, purified 60-fold, that hydrolyzed amino acid (lysine-, and alanine-, or leucine-) p-nitroanilide substrates and leucylglycylglycine, did not hydrolyze [14C]methemoglobin, was completely inhibited by 1 mM bestatin or puromycin (specific inhibitors of leucine aminopeptidase like enzymes), and was unable to influence the sedimentation of the 8S form of the estrogen receptor in sucrose gradients containing dilute Tris buffer. A commercial porcine leucine aminopeptidase, like the calf uterine aminopeptidase, did not convert the 8S estrogen receptor to a 4S form. Evidently, removal of the N-terminal amino acid(s) from the estrogen receptor by exopeptidase action cannot alter the sedimentation of the 8S form of the receptor, or the N-terminal amino acid(s) of the receptor is (are) unaccessible or resistant to exopeptidase activity. The second, a receptor-active protease, is an endopeptidase that did not hydrolyze any of the synthetic amide or peptide substrates tested but did possess [14C]methemoglobin-degrading activity and the ability to convert the 8S estrogen receptor to a modified 4S form in sucrose gradients containing dilute Tris buffer. The modified 4S receptor was separable from the native receptor by DEAE-cellulose chromatography. The endopeptidase did not require Ca2+ for activity, and its chromatographic properties were distinctly different from a previously isolated Ca2+-activated protease. It was inhibited by leupeptin or dipyridyl disulfide, suggesting the presence of a thiol group that is essential for its activity. These data indicate that a decrease in the sedimentation rate of the estrogen receptor in sucrose gradients with low salt or a change in the receptor's elution on DEAE-cellulose chromatography is not related to receptor activation but is produced by the receptor-active protease or other proteases.     

Further characterization of a steroid receptor-active protease from the mature rabbit epididymis

The nucleomyofibrillar fraction of mature rabbit epididymides contains a salt-extractable and leupeptin-sensitive protease that alters the sedimentation coefficient of cytosolic steroid receptors. We refer to this modification as receptor conversion. The substrate used in these studies was cytosolic estrogen receptor obtained from frozen rabbit uteri. The unactivated form of the receptor exists as an oligomer under hypotonic (0.01 M KCl) conditions (S20,w congruent to 9.6, Stokes radius (Rs) congruent to 7.4 nm, Mr congruent to 320,000) and dissociates under hypertonic (0.4 M KCl) conditions to yield the steroid-binding monomer (S20,w congruent to 4.7, Rs congruent to 5.1 nm, Mr congruent to 104,000). According to analysis under hypotonic conditions, the epididymal protease disrupts the oligomeric architecture of the receptor and reduces the size of the steroid-binding monomer (S20,w congruent to 3.2, Rs congruent to 3.0 nm, Mr congruent to 42,000). The epididymal protease had no detectable effect on the structure of the proteins used as standards for the ultracentrifugal or gel filtration analyses. Although inhibited by leupeptin, the epididymal enzyme is not a typical thiol protease since it was unaffected by thiol-blocking agents (iodoacetamide and N-ethylmaleimide), and was partially inhibited by thiol-reducing agents (monothioglycerol and dithiothreitol). Calcium and magnesium ions alone, or in combination with ATP, had no effect on the activity of the protease. However, both cations selectively suppressed recovery of the oligomeric receptor form. These results, in conjunction with those from previous studies, serve to distinguish the epididymal protease from receptor-active proteases described in extracts of other animal tissues. Molybdate, at a concentration of 50 mM, blocked receptor conversion. The ability of the receptor to be stabilized by molybdate was lost following conversion. Finally, the epididymal protease appears to remove a portion of the estrogen receptor that is necessary for nucleotide-binding.     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

A protease acting on the estrogen receptor may modify its action in the adult rabbit epididymis

We have previously shown that the cytosolic estrogen receptor in adult rabbit epididymides sediments as an congruent to 3 S species on sucrose gradients containing 0.01 M KCl while that from immature rabbit epididymides sediments at congruent to 9 S. This age-dependent decrease in sedimentation coefficient is attributable to the appearance of a leupeptin-sensitive protease as the animals mature. We now show that if adult epididymides are homogenized in buffer containing leupeptin, the 9 S receptor can be demonstrated, indicating inhibition of receptor degradation. In vitro nuclear uptake studies conducted in the absence of leupeptin indicated that the proteolyzed receptor was not an efficient nuclear binder. When leupeptin was present, nuclear uptake increased 6-fold and it was accompanied by depletion of receptor from the cytosol. Binding of the receptor to nuclei was specific since it could be inhibited by unlabeled estrogens but not by unlabeled 5 alpha-dihydrotestosterone or progesterone. In vitro mixing experiments indicated that the proteolytic activity was associated with the crude nuclear fraction since, in the absence of leupeptin, they had reduced ability to bind estrogen receptor present in immature epididymal cytosol. Specific in vivo binding of [3H]estradiol by adult and immature rabbit epididymides could be demonstrated. The time course of in vivo binding of [3H]estradiol by adult rabbit epididymal nuclei indicated maximum binding (70 fmol/g tissue) at 30 min following injection. By 60 min, the amount of binding had decreased to about 25 fmol. The accessory sex organs, which do not contain the protease, also exhibited maximum binding (150 fmol/g tissue) at 30 min. However, at the 60 min period binding was still about 140 fmol. Processing the tissues in buffers containing leupeptin had no effect on the results obtained. These results are interpreted to indicate that the presence of the protease decreases nuclear binding of the estrogen receptor and shortens nuclear occupancy. This combination of factors may be responsible for the decrease in estrogen action in the adult rabbit epididymis.     

Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor

It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.     

High molecular weight glycosylated protease in calf brain. Purification and partial characterization

A high molecular weight protease has been purified to homogeneity from calf brain cytosol. The purification procedure involves ammonium sulfate fractionation of the cytosol followed by chromatography on DEAE-Sephacel, hydroxylapatite, concanavalin A-Sepharose 4B and Sephacryl S-300. The molecular weight of the native protease was estimated to be Mr = 465,000 by high pressure liquid chromatography. It is composed of a closely moving doublet of Mr = 165,000 and 155,000, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It degrades [methyl-14C] alpha-casein with a broad pH optimum of 6.8-8.5. [methyl-14C]bovine serum albumin and 125I-bovine serum albumin are hydrolyzed to the same extent as [methyl-14C]alpha-casein, whereas [methyl-14C]methemoglobin is hydrolyzed to half the extent of [methyl-14C] alpha-casein. Divalent cations, nucleotides, and known protease inhibitors (phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, iodoacetic acid, N-ethylmaleimide, leupeptin, antipain, pepstatin, and hemin) have no effect on the activity of the protease. The protease is glycosylated and appears to aggregate readily. Aggregation may be reversed by treating the protease with certain organic solvents. The protease seems to maintain full activity after heat treatment. Electron microscopic data reveals a spherical structure of 20-nm diameter.     

Characterization of glucocorticoid receptors in S49 mouse lymphoma cells by affinity labeling with [3H]dexamethasone 21-mesylate

Glucocorticoid receptors in wild type and mutant S49 mouse lymphoma cells were affinity labeled with [3H]dexamethasone 21-mesylate and analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of receptors in cytosol from wild type cells and nuclear transfer decreased (nt-) mutants was 97,000 (97 kDa). The molecular weight of receptors in cytosol from nuclear transfer increased (nti) mutants was 48 kDa. The 97 kDa receptor in cytosol from wild type cells was digested by chymotrypsin to a 40 kDa steroid-binding receptor fragment but the 48 kDa receptor in cytosol from nti mutants was resistant to digestion by chymotrypsin. In addition to the 48 kDa receptor, cytosol from nti mutants contained 40 and 18 kDa receptor fragments. Cytosol from the nt- mutants also contained 18 kDa receptor fragments. The 40 and 18 kDa receptor fragments were present in multiple subclones of a nti mutant cell line. Formation of these receptor fragments was not prevented by protease inhibitors and was not increased by extended incubation of cytosol samples. Both 48 and 40 kDa forms of the receptor, but not the 18 kDa form, could be activated and bound by DNA-cellulose.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Isolation and characterization of rat liver nuclear glucocorticoid receptor

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitoneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.     

The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

Estrogen-binding proteins of calf uterus. Purification to homogeneity of receptor from cytosol by affinity chromatography.

The estrogen receptor has been purified to homogeneity from calf uterus cytosol by sequential affinity chromatography by using heparin--Sepharose 4B and 17-hemisuccinyl-17beta-estradiol-ovalbumin--Sepharose 4B. The procedure yields about 1.2 mg of receptor protein from 1 kg of calf uteri, with a recovery of 53%. The receptor protein, as a complex with 17beta-[3H]estradiol, is purified more than 99%. A single band is seen on polyacrylamide gel ectrophoresis under nondenaturing conditions. 17beta-[3H]Estradiol comigrates with the protein band. As computed from the specific activity of radioactive hormone, 64,450 g of purified receptor protein binds 1 mol of 17beta-estradiol. 17beta-[3H]Estradiol bound to the protein is displaced by estrogenic steriods but not by progesterone, testosterone, or cortisone. As judged by chromatography on calibrated Sephadex G-200 columns, the purified receptor is identical with native receptor in crude cytosol: both show a Stokes radius of 6.4 nm. On sucrose gradient in low-salt buffer, the purified receptor sediments at 8 S. On electrophoresis in NaDodSO4 gels, the purified receptor migrates as a single protein band with an apparent molecular weight of 70,000. The sedimentation coefficient measured on sucrose gradients in the presence of chaotropic salts [1 M NaBr or NaSCN (0.1 M)] is 4.2 S. We conclude that the estrogen receptor of cytosol consists of a single subunit weighing about 70,000 daltons and endowed with one estrogen binding site. Under native conditions in cytosol, several subunits associate to form a quaternary structure with a Stokes radius of 6.4 nm.     

Phosphorylation of serine 833 in cytoplasmic domain of low density lipoprotein receptor by a high molecular weight enzyme resembling casein kinase II

A soluble protein kinase that phosphorylates the last serine residue (Ser-833) in the cytoplasmic domain of the low density lipoprotein (LDL) receptor was purified about 1300-fold from the cytosol of bovine adrenal cortex. The LDL receptor kinase shared several properties with casein kinase II: use of either GTP or ATP; phosphorylation of a typical casein kinase II recognition sequence in the LDL receptor (a serine followed by a cluster of three negatively charged amino acids); and inhibition by heparin. The LDL receptor kinase differed from classic casein kinase II in the following respects: its apparent molecular weight on gel filtration was approximately 500,000 as opposed to the usual molecular weight of 130,000 for casein kinase II; its affinity for the LDL receptor (apparent Km approximately 5 nM) was much greater than its affinity for casein (approximately 10 microM); and its activity was inhibited by polylysine, an agent that stimulates casein kinase II. The physiologic role of this unusual kinase, if any, is unknown.     

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.     

ATP mediated receptor interconversion as a model of estrogen action: isolation of the factor which converts the non-estrogen binding form of the receptor to the lower affinity binding form

Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third non-estrogen binding form of the receptor, designated Rnb, has also been identified. These three forms of the receptor are interconvertible and appear to have a common molecular weight of approx. 66,000 under denaturing conditions. Hydroxytamoxifen binds preferentially and with high affinity to Rx (Kd 0.03 nM) and the conversion of Rx to Ry which is mediated by gamma phosphoryl group of ATP is also inhibitable by hydroxytamoxifen. Thus receptor interconversion, which may have general application to hormone action, potentially explains agonist/antagonist activity. The conversion of the non-estrogen binding form of the receptor (Rnb) to the lower affinity receptor (Ry) in chick oviduct cytosol is catalyzed by a reaction requiring the loss of the terminal phosphoryl moiety from ATP. There is a specific requirement for Mg2+. We now describe that ammonium sulfate fractionation of the cytosol allows the separation of the receptor entities from the "activating factor" (Fy) that catalyzes the conversion of Rnb to Ry. In the presence of gamma [32P]-ATP at 30 degrees C the purified non-steroid binding form of the receptor is phosphorylated. Phosphoamino acid analysis using Partisil-10 SAX anion exchange resin demonstrates that a serine is phosphorylated; and quantitation of the phosphorylation is indicative of one phosphoserine/receptor molecule. Treatment of the receptor with the partially purified activating factor to induce estradiol binding causes a dramatic reduction in phosphorylation.     

A cytosol protease from the estrogen-resistant C3H mammary carcinoma increases the affinity of the estrogen receptor for DNA in vitro

We have previously shown, in the estrogen-unresponsive C3H mouse mammary tumor that the affinity of the estrogen receptor (ER) for calf thymus DNA in vitro is four-times higher than that of uterine ER [Baskevitch, P. P., Vignon, F., Bousquet, C. and Rochefort, H. (1983) Cancer Res. 43, 2290]. By mixing cytosols from this tumor and uterus, we describe a tumor factor capable of increasing ER affinity for DNA, as assayed by DNA-cellulose chromatography and saturation studies. The activity of this factor was inhibited by alpha-chymotrypsin-inhibitors such as N-tosylphenylalanylchloromethane and chymostatin. Using the fluorogenic substrate glutarylglycylglycylphenylalanyl-N-naphthylamide, we assayed such a protease in the C3H mammary tumor cytosol. This protease and the factor altering ER-DNA binding were eluted together from chromatography on DEAE-cellulose, AcA 44, and carboline-agarose and were sensitive to the same inhibitors. The partially purified factor decreases the molecular mass of the estrogen receptor as seen by denaturing electrophoresis after covalent labelling of the ER with [3H]tamoxifen aziridine. We suggest that the increase of ER affinity for DNA and the decrease of ER molecular size are due to the same protease with an alpha-chymotrypsin-like specificity.     

Molecular weights of estrogen and androgen binding proteins in the liver of Xenopus laevis.

[3-H]Estradiol-17beta and [3-H]dihydrotestosterone binding proteins in the cytosol fraction of liver from both male and female Xenopus laevis were characterized by electrophoresis on polyacrylamide gels. These binding proteins, which were indistinguishable based upon their mobilities on gels of different acrylamide concentrations, migrated as single components with a molecular weight of 2.0 x 10-4. Separation of native or sodium dodecyl sulfate denatured specific estrogen-binding components on dodecyl sulfate free acrylamide gels gave similar results, i.e., a single species of molecular weight 2.0-2.5 x 10-4. The same molecular weight also was obtained when cytosol was prepared in the presence of either diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride, protease inhibitors. Evidence that the liver components binding either [3-H]estradiol-17-beta or [3-H]dihydrotestosterone were not plasma contaminants was provided by the observation that the plasma sex-steroid binding globulin of Xenopus had a different mobility when separated by polyacrylamide gel electrophoresis.     

Estrogen receptors in the adrenal cortex of the possum (Trichosurus vulpecula)

1. Specific [3H]estradiol binding activity with characteristics of estrogen receptors was found in the cytosols and nuclear extracts of the adrenal cortex proper and special zone of the brushtail possum (Trichosurus vulpecula). 2. The specific estradiol receptor had a sedimentation coefficient on sucrose gradients of approximately 9S and a molecular weight on gel filtration of more than 200,000. The adrenal cortex cytosol binds [3H]estradiol with high affinity (Ka 5.5 X 10(9) M-1), and limited capacity (Bmax 62.7 fmol/mg cytosol prot). In competition experiments with different steroids the receptor showed a high affinity for four estrogens and a very low affinity to androgens, progesterone and cortisol. 3. There was no difference in the affinity and maximum binding capacity of the cytosols from cortex proper in male and female animals, but the binding capacity of the special zone of females was half that of cortex proper. Estradiol receptors were found in the kidney, liver, lung, testis and muscle but only in the adrenal and prostate was the binding capacity relatively high compared with the uterus. 4. The specific binding capacity of [3H]estradiol to cytosols of adrenal cortex at different stages of the estrus cycle and pregnancy was unrelated to that of the uterus. In the adrenal the receptor concentration was lowest at estrus, when uterine concentration was high, while in late pregnancy the binding of adrenal cortex and uterus cytosols was almost the same. 5. The possible physiological significance of the presence of a specific estrogen receptor in male and female possums is discussed.     

Age-dependent changes in the multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver.

The soluble NADP-dependent 17 beta-hydroxysteroid dehydrogenase activity of female rabbit liver increases with the age of the animal, the specific activity of the enzyme in the 56-day-old rabbit being 3 times that of the 28-day-old animal. The increase in activity is accompanied by a change in the molecular heterogeneity of the enzyme. Three forms (enzymes I, II and III) were identified in the liver cytosol of the 56-day-old female rabbit, whereas only one major form (enzyme IIIY) was present in the 28-day-old animal. Peptide maps of the four purified enzymes showed that there were minor differences in structure. The enzyme present in the liver of the 28-day-old rabbit was distinct from the three enzymes of the 56-day-old animal. All of the enzymes exhibited bifunctional activity, having 17 beta-hydroxysteroid dehydrogenase activity towards androgen and oestrogen substrates and 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. The differences in substrate specificity of the enzymes paralleled their differences in structure. The data suggest that one enzyme (enzyme III) may have a special role in steroid metabolism during development in the female rabbit.     

A manganese catalyzed covalent binding of estradiol - 17 beta to cytosol proteins of rabbit uterus.

Studies of the temperature sensitivity of estradiol receptor binding in rabbit uterine cytosol have revealed the existence of an enzyme which catalyzes the covalent binding of estradiol to cytosol proteins. A fraction, prepared by chromatography on Biogel P-200 and incubated at 37 degrees C in the presence of Mn++, exhibited a time-dependent, temperature-sensitive, oxidative binding of estradiol not seen in the crude cytosol preparation. Although the activity of this enzyme was shown to be independent of estradiol binding by the high affinity estrogen receptor, its presence may complicate studies of estrogen receptor action which involve the use of elevated temperatures.