Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dihydrotestosterone (DHT) is essential for sperm maturation within the epididymis, but the roles of estradiol-17 beta (E2) and progesterone (P) in epididymal function are unknown. To identify sites of potential action of these hormones, and any effect of season on their concentrations, specific binding of steroids to receptors in extracts of ram epididymal tissue was quantified in two studies. Tissue was taken from three broad regions of the epididymis (caput, corpus, and cauda; Study 1) or from seven discrete regions of the epididymis (Study 2) in February to May (nonbreeding season; NBS) or late August to October (breeding season; BS). Specific binding of P was not detected. Saturable high-affinity binding sites specific for DHT (Ka = 2.6 x 10(8).M-1) and E2 (Ka = 5.4 x 10(8).M-1) were detected. Binding was not to androgen-binding protein, testosterone-estradiol-binding globulin, or sperm nuclei. There was no regional or seasonal difference in affinity of DHT or E2 binding. In both studies, concentration of DHT-binding sites (fmol/mg protein for low- plus high-salt extracts) was higher (p less than 0.05) in the BS than NBS. In Study 1, mean concentration of DHT-binding sites was higher (p less than 0.05) in the caput than in the corpus and cauda. The more definitive localization possible in Study 2 revealed that concentration of DHT-binding sites was highest in the distal caput, lowest in the proximal cauda (p less than 0.05), and intermediate in other regions. For E2, however, concentration of binding sites was higher (p less than 0.05) in the BS than NBS only in Study 1, and was higher (p less than 0.05) in the cauda or corpus than in the caput epididymidis. In Study 2, the season by region interaction was significant (p less than 0.05); concentration of E2-binding sites was higher in the distal cauda during the NBS. These data support the concept that the central caput through proximal corpus epididymidis are most dependent on androgenic stimulation, whereas distal regions may respond to estrogenic stimulation.
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PMID:Regional and seasonal differences in concentrations of androgen and estrogen receptors in ram epididymal tissue. 340 73

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.
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PMID:Further characterization of a steroid receptor-active protease from the mature rabbit epididymis. 353 65

The nucleomyofibrillar fraction of epididymides from sexually mature rabbits contains a novel leupeptin-sensitive protease that disrupts the oligomeric conformation of cytosolic estrogen and progesterone receptors, and molybdate inhibits this process. In this report we used the AtT-20 cell glucocorticoid receptor as substrate and performed analyses under nondenaturing vs. denaturing conditions to further investigate the effects of the epididymal protease and molybdate on steroid receptor structure. Analysis on low salt sucrose gradients indicated that the protease partially converted the oligomeric (9-10S) glucocorticoid receptor to several more slowly sedimenting forms (3-7S), and this effect was not observed in the presence of molybdate. Paradoxically, gradient analysis under high salt conditions revealed that the protease induced a discrete, quantitative and molybdate-insensitive conversion of the 4-5S steroid-binding subunit to a 3S form. Further studies were done using denaturing polyacrylamide gel electrophoretic analysis of receptor that had been labeled covalently with [3H]dexamethasone 21-mesylate and partially purified by DNA/cellulose chromatography. At 0-4 C, the protease cleaved the steroid-binding subunit (mol wt, 96,900) of the receptor to a single steroid-labeled fragment (mol wt, 42,600). Under these conditions, digestion was complete within 30-60 min and was inhibited by leupeptin, but was unaffected by thiol-reactive reagents or molybdate. The epididymal protease and alpha-chymotrypsin produced steroid-labeled receptor fragments that were indistinguishable in size, shared an epitope recognized by our BuGR-2 monoclonal antibody, and retained DNA-binding activity. Despite the apparent similarity of these two enzymes, they are distinct, since the chymotrypsin-dependent cleavage event was not inhibited by leupeptin. These studies show that the epididymal protease attacks a site on the steroid-binding subunit of glucocorticoid receptors as well as estrogen and progestin receptors. It also appears that the cleavage site is situated close to that most readily attacked by alpha-chymotrypsin. Finally, our data provide independent confirmation of a recent report indicating that molybdate ions interact directly with the cytosolic steroid receptor to stabilize its oligomeric structure even after proteolysis within the steroid-binding subunit.
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PMID:Analysis of the disruptive action of an epididymal protease and the stabilizing influence of molybdate on nondenatured and denatured glucocorticoid receptor. 354 7

Chromatography of aspartate transcarbamoylase from Escherichia coli on agarose-immobilized dyes and alkyl-agaroses of differing carbon length were investigated. The bacterial aspartate transcarbamoylase was bound by Procoin red HE3B-agarose and Cibacron blue F3GA-agarose nearly completely under the conditions chosen relative to other agarose-coupled dyes. The aspartate transcarbamoylase holoenzyme was eluted from the Procion red HE3B-agarose slightly later than from the Cibacron blue F3GA-agarose during salt gradient elution. The catalytic trimer of the enzyme as well as its regulatory dimer were eluted by a lower salt concentration from both dye-agarose gels than the concentration required to elute the holoenzyme. The interaction of the catalytic trimer with the Procion red HE3B-agarose and Cibacron blue F3GA-agarose gels may be a determinant in the holoenzyme being retained on these resins. Of those alkyl-agaroses tested, the ethyl-, propyl- and hexyl-agarose gels bound the majority of aspartate transcarbamoylase activity. Chromatography of aspartate transcarbamoylase on ethyl-agarose found it to be eluted by a low salt concentration. A purification scheme for relatively small amounts of aspartate transcarbamoylase utilizing Procion red HE3B-agarose and ethyl-agarose is presented. This purification scheme is particularly useful for mutant versions of aspartate transcarbamoylase which cannot be purified by literature procedures.
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PMID:Affinity and hydrophobic chromatography of Escherichia coli aspartate transcarbamoylase. 388 51

The nuclear T3 receptor was characterized in two T3-responsive preadipocyte cell lines cloned from the epididymal fat pads of ob/ob adult mice (ob 17 cells) and their lean counterpart (HGFu cells). Isolated nuclei from confluent or differentiating cells bound [125I]T3 to one class of high affinity sites exhibiting kinetic properties, stereospecificity, and salt extractibility of the nuclear T3 receptor. The solubilized T3 binding sites behave like the hepatic nuclear T3 receptor considering physicochemical and DNA binding properties. At confluence, no significant difference could be detected in equilibrium apparent affinity constant (Ka) and maximum binding capacity (MBC) for T3 whether nuclei were prepared from cells originating from ob/ob mice [Ka: 1.7 +/- (SE) 0.5 X 10(10) M-1; MBC: 432 +/- 29 fmol/mg DNA] or from lean mice (Ka: 1.6 +/- 0.2 X 10(10) M-1; MBC: 487 +/- 39 fmol/mg DNA). MBC values were in the range found in several T3-responsive tissues. This suggests that the primary defect in ob/ob mice is probably not at the level of the nuclear T3 receptor. Furthermore, during differentiation into adipose cells in both cell series, and roughly paralleling the amplifying effect of T3 on several lipogenic enzymes in the course of their development, the nuclear T3 receptor concentration significantly increased, attaining about twice the initial values after completion of the differentiation without any significant change in the affinity for T3.
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PMID:Nuclear triiodothyronine receptor in differentiating preadipocytes cloned from obese and lean mice. 631 54

A number of dye-ligand adsorbents have been examined for purifying and characterizing 1,25-dihydroxyvitamin D3-receptor complexes from intestines of vitamin D3-deficient chickens. In particular, several triazinyl dyes--Cibacron blue F3GA, Procion red HE3B, and Green A dye, immobilized to agarose via an ether linkage--retain specifically bound 1,25-dihydroxyvitamin D3-receptor complexes formed at 0-4 degrees which are eluted at high salt concentrations. Moreover, receptor binding to these dye-ligand matrices occurs in the presence and absence of sterol. At least for Cibacron blue, the strength of receptor binding depends critically on the method of dye coupling to matrix. The concentration of KCl required for elution of receptor from the triazine ether-linked matrix is greater than coupling through the amine of the anthraquinone via a 10-atom spacer arm approximately equal to coupling through the amine of the anthraquinone via an isourea bond greater than Cibacron blue dextran. Data are presented which demonstrate that sterol-receptor complexes formed at 25 degrees have reduced affinity for dye-ligands when compared with sterol-receptor complexes formed at 0-4 degrees. It is suggested that this finding is related to proteolytic alterations of the receptor, since limited digestion with trypsin can mimic this phenomenon and several protease inhibitors can reduce the thermal-induced alterations. Biospecific elution of receptor is demonstrated using synthetic polyribonucleotides. Preference for polyguanylic and polyinosinic acid is observed over several other polyribonucleotides and mononucleotides. The data in this study, viewed collectively, suggest that there is a specific interaction between the polynucleotide domain of the 1,25-dihydroxyvitamin D3-receptor and several triazinyl dye-ligands. It is concluded that these dye-ligands should prove to be of considerable interest for facile chromatography to purify and characterize this receptor.
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PMID:Dye-ligand interactions with 1,25-dihydroxyvitamin D3-receptor complexes from chicken intestine. 632 53

Insulin from an elasmobranch, the spiny dogfish (Squalus acanthias) has been purified to near homogeneity by means of acid-ethanol extraction and salt precipitation. The amino acid sequences of the performic-acid-oxidised A and B chains have been determined and exhibit some unusual features. The A chain contains a total of 22 amino acids; only the insulin from coypu (a member of the Rodentia suborder, Hystricomorpha), has previously been reported to contain an extension past the A21 asparagine. The B10 histidine, which is involved in the formation of the insulin hexamers in higher vertebrates through the co-ordination of zinc, is present in this elasmobranch insulin. Several substitutions relative to bovine insulin occur in the proposed receptor binding region (A5Gln leads to His, B21Glu leads to Pro, B22Arg leads to Lys, B25Phe leads to Tyr). In spite of these substitutions, the maximal response in the rat epididymal fat cell assay is the same for bovine and dogfish insulins; the concentration required to produce the half-maximal response is, however, approximately threefold greater for dogfish insulin than that of bovine insulin. The use of interactive computer graphics model-building predicts that the dogfish insulin can attain a three-dimensional structure very similar to that of bovine insulin; circular dichroic spectra are presented which support the model-building studies.
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PMID:Dogfish insulin. Primary structure, conformation and biological properties of an elasmobranchial insulin. 635 61

Tricyclic antidepressants appeared to be without effect, except for desipramine which significantly decreased whiplash motility after spermatozoa were added to eggs, and clomipramine which decreased motility and whiplash motility in epididymal sperm suspensions after pretreatment of males. Mianserin and viloxazine were also without effect, but nomifensine significantly decreased sperm motility and whiplash motility and inhibited egg penetration almost completely. After 3 h preincubation with 0.75 mmol nomifensine hydrogen maleate/l, 2/181 and 0/256 eggs were penetrated in two separate series of experiments. Control groups in these series gave medians of 90-100% penetration by 4.5-5.5 h after spermatozoa and eggs were mixed. Maleic acid had a similar effect (1/253 eggs penetrated) whilst nomifensine hydrochloride was inactive, suggesting that the effect was due to the maleate moiety of the original nomifensine hydrogen maleate salt used.
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PMID:Effect of antidepressant drugs on the in-vitro egg-penetrating ability of golden hamster epididymal spermatozoa. 668 37

A study was made of unoccupied androgen binding sites in the nuclei of ventral prostate glands of male rats. They were measured at 0 degrees C by comparing specific binding of 1 nM [3H]DHT to salt extract of purified nuclei during the first hour with specific binding during both hours. This method was dependent upon demonstrated completion of uptake into unoccupied binding sites within the first hour and linearity of exchange with occupied binding sites during both hours. Unoccupied binding sites were not artefactual. They did not increase if tissue concentration was diluted prior to homogenization, while they decreased if homogenization was delayed after the tissue was minced. They could be occupied, both in vitro (if precharged with at least 1 nM unlabeled DHT) or in vivo, by administering testosterone propionate subcutaneously or by infusing testosterone into the jugular vein. Exposure to a high concentration of unoccupied prostatic cytosolic binding sites (608.4 fmol from castrated rats) as compared to low concentration (29.3 fmol from intact rats) during homogenization had little effect upon nuclear unoccupied binding site concentrations (2.16 fmol/mg DNA vs 2.41 fmol/mg DNA, respectively). In individual rats, concentration of unoccupied nuclear androgen binding sites was 4.61 +/- 1.05 fmol/mg DNA, while total binding site concentration (measured with 10 nM [3H]DHT for 24h at 12 degrees C) was 866 +/- 103 fmol/mg DNA. Unoccupied nuclear binding sites reached their highest concentration in animals 4 months old (15.09 fmol/mg DNA) when animals 21 days through 720 days of age were studied. By use of association and dissociation rates of binding, it was determined that the apparent Kd of nuclear binding sites was 1.11 X 10(-12) M. There were no observed differences between unoccupied and occupied binding sites in steroid specificity or in sedimentation rate in an 8-24% glycerol density gradient. Although no physiological importance can be attributed as yet to unoccupied nuclear androgen binding sites in the prostate, they do provide a convenient comparison with putative androgen binding sites in the nuclei of testicular and epididymal germ cells.
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PMID:Nuclear androgen binding sites in the male rat. I. Unoccupied sites in the prostate. 674 44

Nuclear androgen binding sites were examined in late spermatids (stages 12-19) which resisted sonication of homogenized testes of mature male rats. The measurement of unoccupied binding sites in salt extract of purified spermatid heads by nuclear exchange at -10 degrees C was developed and validated. As in the prostate, unoccupied nuclear androgen binding sites in sonicated testes were in low concentration, were not artefactual, and could be occupied both in vivo and in vitro by exogenous androgens, and uniquely in hemicastrated rats by endogenously compensated androgens in the remaining testis. The properties of occupied binding sites in salt extract of purified spermatid heads (measured by nuclear exchange at 4 degrees C for 48 or more hours with 5 nM [3H]dihydrotestosterone) were almost identical to those of occupied binding sites in nuclei of the ventral prostate, except for their concentration. However, levels of specific binding activity approaching 50 fmol/mg DNA could be expected in salt extract of spermatid pellets, by use of a sulfhydryl reducing agent (dithiothreitol) prior to salt extraction, a protease inhibitor (phenylmethylsulfonyl fluoride) in all buffers, and optimization of the sonication protocol. Nuclear androgen binding sites of sonicated epididymal spermatozoa, collected by retrograde perfusion of the cauda epididymidis, were found to be completely salt-resistant. These binding proteins could be extracted by 0.4 M KCl if dithiothreitol and dihydrotestosterone were incorporated into the sonication buffer, if phenylmethylsulfonyl fluoride was added to all buffers, and if the purified epididymal sperm pellet was treated with sarkosyl, a non-ionic detergent, just before salt extraction. The salt extract of epididymal spermatozoa which were treated as described above contained two binding components: a soluble form which was eluted from hydroxylapatite by increasing concentrations of phosphate buffers, and a non-soluble form, free of DNA, which remained in the hydroxylapatite column, and which contained most of the androgen binding sites. Affinity (Kd) of dihydrotestosterone to the soluble and insoluble fractions of the steroid-binding protein complex was determined to be 0.7 and 0.1 nM, respectively. Salt-resistance of binding proteins in germ cells was shown to develop significantly in the last stages of spermiogenesis.
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PMID:Nuclear androgen binding sites in the male rat. III. Late spermatids and spermatozoa in the testis, with an introduction to epididymal spermatozoa. 674 45


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