Properties of Crystalline 3β-Hydroxysteroid Dehydrogenase from Pseudomonas putida

A crystalline 3α-hydroxysteroid: NAD⁺-oxidoreductase (EC 1 1.1.50) which had been obtained from the cell-free extracts of Pseudomonas putida NRRL B-11064 in the presence of added polyethylene glycol, was found to be a native monomer form with a specific activity of 63.0 and a molecular weight of 45,000. Isoelectric focusing exhibited the enzyme to be composed of two isoenzymes: one major part focusing at pH 4.75 and a minor part focusing at pH 5.10. Whereas the enzyme was changed from the monomeric form to a dimeric one with a considerable decrease in the specific activity during the course of crystallization in the absence of the added polyethylene glycol.

The enzyme showed an absolute specificity with regard to 3α-hydroxyl group besides a high requirement for cis A: B fusion of steroids. Typical substrates are cholic acid (Km = 1.33 × 10^?5 m), deoxycholic acid, chenodeoxycholic acid, 3α-hydroxy-12-keto-9,11-cholanoic acid, and etiocholan-3α-ol-17-one. Conjugated bile acids such as taurocholic acid and glycocholic acid are also rapidly oxidized. The pH optima for oxidation of cholic acid and reduction of etiocholan-3,17-dione were 11.5 and 7.0, respectively. The enzyme could be employed for the sensitive and specific assay of bile acids.     

3α-Hydroxysteroid Dehydrogenase in Animal and Human Tissues

This review analyzes data on the biological role of 3-hydroxysteroid dehydrogenase (3-HSD) in animal and human tissues and describes its main characteristics, mechanism of action, and regulation of activity. Based on published data, a scheme for the actions of androgen, progestin, and glucocorticoids involving the participation of 3-HSD is proposed. According to this scheme, in the mechanism of steroid action 3-HSD not only regulates the concentration of the main effector androgen, 5-dihydrotestosterone, in target cells, but also switches androgen, progestin, and glucocorticosteroid genomic activity to non-genomic activity.     

Interaction of Cytotoxic Bicyclic Peptides, Theonellamides A and F, with Glutamate Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase IV

Theonellamide A, a bicyclic peptide isolated from a Theonella sponge, was fixed on hydrazide-containing gel beads and screened for its binding proteins from rabbit liver tissues. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that two major proteins of 80 kDa and 55 kDa interacted with theonellamide A. The interaction between theonellamide A and two proteins was confirmed by competition experiments in which these two proteins failed to bind to theonellamide A–conjugated gel beads in the presence of theonellamide A or F. Amino-terminal amino acid sequence analysis of peptide fragments derived from the binding proteins by lysylendopeptidase digestion demonstrated that the 80-kDa and 55-kDa proteins were 17β-hydroxysteroid dehydrogenase IV and glutamate dehydrogenase, respectively. In an in vitro assay system, amination of α-ketoglutarate by glutamate dehydrogenase was activated with theonellamide F, although this effect was weaker than that with adenosine diphosphate, a well-known activator. Received October 15, 1999; accepted January 4, 2000.     

Purification,Crystallization and Some Properties of β-Galactosidase from Saccharomyces fragilis

Crystalline β-galactosidase was prepared from the cell extract of Saccharomyces fragilis KY5463, by procedures including protamine sulfate treatment and DEAE-cellulose, hydroxylapatite and DEAE-Sephadex column chromatographies. Crystals were formed when solid ammonium sulfate was added to solutions of the purified enzyme. This procedure resulted in a 55-fold purification with an over-all yield of l5.4%. The crystalline enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis.

The sedimentation coefficient, , was determined to be 10.0 S. The molecular weight was estimated to be approximately 203,000 by the sedimentation equilibrium method of Yphantis. Electrolysis with carrier ampholytes revealed that this enzyme has an isoelectric point at around pH 4.4.

The enzyme was activated by K⁺ in addition to bivalent cations, such as Mn²⁺, Mg^2? and Co²⁺. The Km values for o-NPG and lactose were 4.0×10^?3m and 21.0×10^?3m, respectively. The enzyme is sulfhydryl dependent and was completely inactivated by mercuric ions or p-chloromercuribenzoate.     

Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.     

Production,Purification, and Characterization of α-Amylase from Solventogenic Clostridium sp. BOH3

A solventogenic strain of Clostridium sp. BOH3 produces extracellular α-amylase (7.15 U/mg protein) in reinforced clostridial medium supplemented with sugarcane bagasse hydrolysate (1 % w/v) and a small amount of starch (0.1 % w/v), which is essential for the expression of α-amylase. In the presence of α-amylase, BOH3 utilizes starch directly without any pretreatment and produces butanol almost equivalent (～90 %) to the production of butanol from glucose. α-Amylase can be purified from culture supernatant by using one-step weak anion exchange chromatography with a yield of 43 %. In peptide fingerprinting analysis, this enzyme shows homology with α-amylase produced by Clostridium acetobutylicum ATCC824. However, the molecular weight is 54 kDa, which is smaller than α-amylase of ATCC824 (84 kDa). This enzyme has optimum temperature at 45–50 °C and optimum pH at 4.5–5.5. Under this condition, the enzyme activity is 91.32 U/mg protein, and its K _m and V _max values are 1.71?±?0.02 mg/ml and 96.13?±?0.15 μmol/min/mg protein, respectively. Activity of this α-amylase can be enhanced (>1.5 times) by addition of Ca²⁺ and Co²⁺ and its activity can be maintained at an acidic pH (pH 3–5) for about 24 h. These unique characteristics suggest that this enzyme can be used for saccharification of starch for production of biofuel in one pot.     

Purification,Characterization, and Crystallization of Single Molecular Species of β-Conglycinin from Soybean Seeds

Four major molecular species of β-conglycinin, α₃, α_2β, αβ₂, and β₃, were isolated and purified from seeds of an α' subunit-deficient strain of soybeans (Glycine max). All components were found to be homogeneous by high pressure liquid chromatography, SDS-polyacrylamide gel electrophoresis, and amino acid and amino terminal sequence analyses. The amino acid compositions of the α₃ and β₃ components agreed fairly well with the compositions deduced from the cDNA sequences, and all of the components were highly glycosylated. The α₃ and β₃ components were compared regarding their secondary structures. The secondary structure of the α₃ component deduced from CD measurements showed a higher α-helix content than that of the β₃ component. The β₃ component was crystallized by decreasing the ionic strength from 0.5 to 0.14 in phosphate buffer, pH 7.3, and the crystals grew to a size (1.0 mm × 0.2 mm × 0.2 mm) suitable for X-ray crystallographic analysis. A preliminary X-ray analysis showed that the crystal belonged to an orthorhombic crystal system having the space group P2₁2₁2₁ and unit cell dimensions of a = 185.1 Å, b = 107.9 Å, and c = 97.6 Å.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

Molecular Framework of Steroid/Retinoid Discrimination in 17β-Hydroxysteroid Dehydrogenase Type 1 and Photoreceptor-associated Retinol Dehydrogenase

Steroids and retinoids are signaling molecules that control a variety of physiological processes. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reduction of estrone to estradiol, supplying biologically active estrogen-regulating sex-specific differentiation. Photoreceptor-associated retinol dehydrogenase (prRDH) is evolutionarily closely related to 17β-HSD1 but reduces all-trans retinal to all-trans retinol, contributing to rhodopsin regeneration in the visual cycle. Sequence alignment revealed a new enzyme-specific conserved amino acid close to the active site: methionine (position 144 in human enzyme) in prRDH and glycine (position 145) in 17β-HSD1. We investigated the role of this residue in substrate discrimination in human and zebrafish enzymes. Both recombinant enzymes were expressed in HEK 293 cells followed by normalization of expression by semiquantitative Western blots. Changing of the prRDH-specific methionine to glycine resulted in a gain of function: the mutants now catalyzed the reduction of estrone and all-trans retinal. Human and zebrafish wild-type 17β-HSD1s efficiently catalyzed the reduction of all-trans retinal to its alcohol. Exchange of glycine for methionine increased the catalytic activity of 17β-HSD1 toward all-trans retinal in zebrafish but not in the human enzyme, in which the opposite effect was observed. Molecular modeling showed that the zebrafish 17β-HSD1 substrate-binding pocket is similar to that of prRDH and methionine insertion benefits all-trans retinal reduction. In contrast, in human 17β-HSD1, the insertion of the bulky methionine causes a disruption of substrate-binding site. We demonstrate for the first time the role of a single amino acid in the evolution of these functionally diverse enzymes and suggest new physiological functions for 17β-HSD1 in retinoid metabolism. This has implications for the validation of inhibitors of 17β-HSD1 developed for cancer treatment.     

Production of two monomer structures containing medium-chain-length polyhydroxyalkanoates by β-oxidation-impaired mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) from fatty acids. When gene encoding 3-hydroxyacyl-CoA dehydrogenase which catalyzes long-chain-3-hydroxyacyl-CoA to 3-ketoacyl-CoA, was partially or completely deleted in P. putida KTOY08, the PHA accumulated was shown to contain only two different monomer structures dominated by a monomer of the same chain length as that of the fatty acids fed and another monomer two carbon atoms shorter. Among the PHA copolymers, P(44% 3HD-co-3HDD) containing 44% 3HD and 56% 3HDD was demonstrated to have a melting temperature T_m, an apparent heat of fusion △H_m and a Young’s modulus E of 75 °C, 51 J g^?1 and 2.0 MPa, respectively, the highest among all the MCL PHA studied.     

Interactions of 17β-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease

The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (k_a 2.0?×?10⁵ M¹s^?1, k_d 5.8?×?10⁴ s^?1, and K_D 3.5?×?10^–10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10–cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10–cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10–cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

     

Metabolism of Androstenone, 17β-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3β-Hydroxysteroid Dehydrogenase in This Process

Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5∶1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.     

Efficient Production and Purification of Extracellular 1,2-α-L-Fucosidase of Bacillus sp. K40T

When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-α-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5–7.0 and was stable at pH 6.0–9.0. The enzyme hydrolyzed the α(1 → 2)-L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 〈O-α-L-fucose-(1 → 2)-O-β-D-galactose-(1 → 3)-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze α(1 → 3)-, α-(1 → 4)- and α-(1 → 6)-L-fucosidic linkages in LNF-III 〈O-β-D-galactose-(1 → 4)[O-α-L-fucose-(1 → 3)-]-N-acetyl-O-β-D-glucosamine-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉, LNF-II 〈O-β-D-galactose-(1 → 3)[O-α-L-fucose-(1 → 4)-]-N-acetyl-O-β-D-galactose-(1 → 3)-O-β-D-galactose-(1 → 4)-D-glucose〉 or 6-O-α-L-fucopyranosyl-N-acetylglucosamine.     

Biosynthesis of poly(3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by β-oxidation pathway inhibited Pseudomonas putida

Pseudomonas putida KT2442 produces medium-chain-length polyhydroxyalkanoates consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxytetradecanoate (3HTD) from relevant fatty acids. P. puitda KT2442 was found to contain key fatty acid degradation enzymes encoded by genes PP2136, PP2137 (fadB and fadA) and PP2214, PP2215 (fadB2x and fadAx), respectively. In this study, the above enzymes and other important fatty acid degradation enzymes, including 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA dehydrogenase encoded by genes PP2047 and PP2048, respectively, were studied for their effects on PHA structures. Mutant P. puitda KTQQ20 was constructed by knocking out the above six genes and also 3-hydroxyacyl-CoA-acyl carrier protein transferase encoded by PhaG, leading to a significant reduction of fatty acid β-oxidation activity. Therefore, P. puitda KTQQ20 synthesized homopolymer poly-3-hydroxydecanoate (PHD) or P(3HD-co-84mol% 3HDD), when grown on decanoic acid or dodecanoic acid. Melting temperatures of PHD and P(3HD-co-84mol% 3HDD) were 72 and 78 °C, respectively. Thermal and mechanical properties of PHD and P(3HD-co-84mol% 3HDD) were much better as compared with an mcl-PHA, consisting of lower content of C10 or C12 monomers. For the first time, it was shown that homopolymer PHD and 3HDD monomers dominating PHA could be synthesized by β-oxidation inhibiting P. putida grown on relevant carbon sources.     

Purification of ecdysone oxidase and 3-dehydroecdysone 3α-reductase from the cotton leafworm,Spodoptera littoralis

The 3-epimerization of ecdysteroids (insect moulting hormones) is an inactivation pathway of the hormones that has been reported to occur in midgut cytosol of Lepidoptera. The pathway involves ecdysone oxidase-catalysed conversion of ecdysone into 3-dehydroecdysone, which is then irreversibly reduced to 3-epiecdysone by 3DE 3α-reductase. In this study, ecdysone oxidase and 3DE 3α-reductase from the cotton leafworm, S. littoralis, have been purified by extensive chromatography together with electrophoresis on native gels. Gel filtration suggested that the native ecdysone oxidase might be a trimer with apparent molecular mass of approximately 190 kDa, since the apparent molecular mass of the oxidase subunit was determined to be 64 kDa by SDS-PAGE. Two forms of 3DE 3α-reductase were observed during the purification, the 26 kDa form reductase has been purified to homogeneity and the second form of the reductase identified as a 51 kDa protein. The former reductase may be a trimer with apparent molecular mass of 76 kDa, whilst the latter was suggested to be a monomer by gel filtration. Chromatographic behaviour suggested that the 26 kDa form of the reductase has a lower pI value and a higher degree of hydrophobicity than that of the 51 kDa reductase. Substrate specificity and the tissue distribution of these enzymes are discussed.     

Expression,Purification, and Characterization of a Human Recombinant 17β-Hydroxysteroid Dehydrogenase Type 1 in <Emphasis Type=Italic>Escherichia coli</Emphasis>

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) catalyzes the reaction of estrone with NADPH to form estradiol and NADP⁺, thereby regulating the biological activity of sex steroid hormones in a variety of tissues. Here, we present an efficient method for expressing and purifying human 17β-HSD1 from Escherichia coli. The expression vector pET28a/17β-HSD1 was constructed and transformed into Escherichia coli BL21(DE3) cells. We found that the active enzyme can be obtained by inducing 17β-HSD1 expression at 0.25 mM IPTG, 13°C for overnight. The protein is purified by single step Ni–NTA affinity chromatography and yields 2.8 mg/L of culture. The kinetic study shows V/E _t of (1.21 ± 0.05) × 10⁻²/s and K _estradiol of 0.8 μM in the oxidation of estradiol with NADP⁺ as cofactor at pH 9.3. The new bacterial expression system for recombinant 17β-HSD1 is useful for the easy purification of large amounts and will facilitate the functional study of this enzyme.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

3α-Hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

Summary The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3-hydroxysteroid and the 3-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6.During the entire estrous cycle, 3-hydroxysteroid dehydrogenase and 3-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.This investigation was supported by the Deutsche Forschungsgemeinschaft