Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.
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PMID:Effects of proteases and protease inhibitors on the 4.5 S and 8 S androgen receptor. 44 15

We have investigated the ability of dehydroepiandrosterone (DHEA) to alter the production of interleukin-2 (IL-2) and to bind to a specific binding complex in antiCD3 epsilon activated T cells. Binding activity correlated with the presence of a specific DHEA binding complex in the cytosol and nuclei of DHEA-responsive T-cell hybridomas, as well as in CD4+ and CD8+ cells isolated from peripheral lymph nodes of normal mice. Scatchard analysis determined that intact lymphocytes and cytosolic fractions contained high affinity binding for [3H]DHEA (approx. 2.6 nM) with 1000-7000 binding sites existing per cell. Five of the T-cell hybridomas tested both responded to DHEA treatment with increased production of IL-2 and also contained specific high affinity [3H]DHEA binding. Four additional T-cell hybridomas were found to contain no specific [3H]DHEA binding and were also unresponsive to DHEA influences on IL-2 production. Sucrose density gradients demonstrated a 3-4s [3H]DHEA binding complex in high salt and a 7-8s binding complex in low salt. Specific binding was inhibited by preincubation of the cytosol fractions with either trypsin or chymotrypsin, or by heating to 60 degrees C for 1 h (less than 15% of control). [3H]DHEA binding was unaffected by preincubation of the cytosol fractions with ribonuclease, deoxyribonuclease, or phospholipase A. The DHEA-protein complexes bound to DNA-cellulose with the amount of binding being slightly increased by preincubation at 25 degrees C as compared to 4 degrees C. As expected, [3H]DHEA binding was inhibited by the addition of unlabeled DHEA, but was also modestly inhibited by dihydrotestosterone and cortisol. Binding of DHEA was unaffected by progesterone, dexamethasone, estradiol, androsterone, DHEAS, and beta-etiocholanolone at all concentrations tested. DHEA was incapable of inhibiting the binding of [3H]DHT to the androgen receptor or [3H]dexamethasone to the glucocorticoid receptor. Collectively, these findings suggest that murine T cells contain a specific DHEA receptor. We believe that DHEA is a steroid hormone that is directly involved in the regulation of IL-2 production by both normal and some T-cell hybridomas.
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PMID:The presence of a dehydroepiandrosterone-specific receptor binding complex in murine T cells. 135 1

PSA is a 34-kd 240-amino acid glycoprotein produced by the prostatic epithelial cells. It is a member of the glandular kallikrein gene family and has a high sequence homology with human glandular kallikrein (hGK-1). PSA is a serine protease and has chymotrypsin-, trypsin-, and esterase-like activities. It is secreted into the seminal fluid where it degrades two seminal vesicle proteins that are important components of the semen coagulum, thus playing an important role in semen liquefaction. The production of PSA protein appears to be under the control of circulating androgens acting through the androgen receptor. Therefore, the significance of a low serum PSA value in a patient who has undergone previous antiandrogen therapy may not be the same as that for a patient who has not received endocrine treatment.
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PMID:Prostate-specific antigen and prostatic acid phosphatase: biomolecular and physiologic characteristics. 171 6

The DNA-binding form of the calf uterine androgen receptor (AR) was subjected to limited protease digestion using chymotrypsin, trypsin and a rat prostate cytosol protease. The properties of the generated polypeptide fragments were identified and compared with those of the intact AR. Physicochemical characterization was achieved through sedimentation analysis, gel filtration chromatography and DEAE anion exchange chromatography. Intactness of functional binding domains was evaluated by measuring the retention of steroid- and DNA-binding capacity. Under non-denaturing conditions the intact AR is a highly asymmetrical molecule with a Stokes radius (RS) of 45A, a sedimentation coefficient of 4.3S and a relative molecular mass of 80,000 daltons. This form of AR has an intrinsic binding affinity for DNA and was eluted from DNA-cellulose with 9 mM MgCl2. Chymotrypsin produced a more globular polypeptide (RS: 31A; 3.1S; 41,000 daltons) with a decreased net negative charge. This fragment also displayed DNA-binding affinity but required a higher concentration of MgCl2 (14 mM) for DNA-cellulose elution, indicating an increased affinity for DNA. The observed reduction in molecular size upon chymotrypsin treatment was confirmed when analysed by SDS-polyacrylamide gel electrophoresis after covalently labelling of the AR with [3H]R1881. Rat prostate cytosol contains a protease which is very active in generating an AR polypeptide with an increased affinity for DNA, without changing the AR net negative charge (RS: 33A; 3.7S; 51,000 daltons). The specificity of this protease remained unknown since none of a large number of inhibitors was able to inactivate this enzyme. The fragment generated is different from that obtained with chymotrypsin since significant differences in size as well as in charge were measured. Trypsin treatment generated a much smaller polypeptide (RS: 25A; 2.9S; 30,000 daltons) which had lost its DNA-binding capacity, but not its steroid binding site. This form probably represents the so-called meroreceptor. When intact AR was treated sequentially with prostate cytosol and trypsin, a polypeptide fragment with identical properties was obtained, indicating the spatial separation of two of the proteolytic cleavage sites. These studies provide evidence for the distinct nature of the molecular domains for androgen and DNA interaction on the calf uterine AR.
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PMID:Analysis of steroid- and DNA-binding domains of the calf uterine androgen receptor by limited proteolysis. 330 38

Available androgen binding to soluble proteins from the cytosol of human endometrium was studied using the dextran coated charcoal adsorption method and sucrose density centrifugation analysis. Specific binding of [3H]-5 alpha-dihydrotestosterone ([3H]-DHT) was observed with both methods. The apparent dissociation constant (Kd), for DHT binding is 1.3 +/- 0.2 (SEM) nM and the binding capacity 177 +/- 42 (SEM) fmol/mg protein. Sucrose density ultracentrifugation identifies specific [3H]-DHT binding that sediments at 4S and 8S. The stability of the androgen receptor in human endometrium is increased by the addition of 10% glycerol to the homogenization buffer. The addition of trypsin or pronase and heating at 60 degrees C reduces specific binding which demonstrates that the specific [3H]-DHT binder is a protein. The uptake of [3H] DHT in endometrial tissue minces indicated that 20% of the bound radioactivity was nuclear. Steroid specificity suggests that the binding protein from the uterus is specific for androgens. These observations indicate that androgen binding protein in the human uterus has the characteristics of the androgen receptor.
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PMID:The androgen receptor of the human endometrium. 358 78

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.
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PMID:DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate. 369 93

The presence of a macromolecule which binds androgen with a high affinity and a low capacity was demonstrated in the cytosol of the lacrimal glands of male and female rats. Evidence was found that this macromolecule was a protein by treatment with protease, trypsin or heat. A specific 8-8.5 S peak was obtained in both sexes by glycerol gradient centrifugation in low salt condition, whereas a specific 5.2 S peak was found in high salt condition. This protein could bind to DNA-cellulose after treatment of androgen-cytosol complexes by warming (25 degrees C 15 min) or exposure under high salt (0.4 M KCl). These results suggested that this protein was an androgen receptor.
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PMID:Demonstration and characterization of cytosol androgen receptor in rat exorbital lacrimal gland. 387 41

The physical properties of two types of androgen-binding sites in prostatic nuclei were compared and found to be identical. The first type was released from chromatin by micrococcal nuclease digestion and solution in 0.6 M NaCl; the second resisted such treatment and remained associated with nuclear structures. After in vivo administration of [1,2-3H]testosterone to 24-h castrated rats and sonication of purified nuclei, 90% of the nuclear radioactivity was extracted with nuclease/salt treatment and was found by sucrose density gradient analysis to be associated with a 3 S androgen receptor. If sonication was omitted, 50 to 60% of the nuclear radioactivity was recovered in the nuclease/salt-resistant pellets or bound to nuclear matrices. Mild digestion of either of these particulate fractions with trypsin resulted in the release of a 3 S androgen receptor. After in vitro isotope-exchange labeling with [1,2-3H]dihydrotestosterone, the sedimentation coefficient, steroid specificity, and dissociation constant of the androgen receptors released by trypsin digestion of nuclease/salt-resistant pellets or nuclear matrices were similar to those of the receptors extracted by nuclease/salt treatment. These results indicate first, that all androgen-binding sites in prostatic nuclei can be released, either with nuclease/salt or trypsin digestion procedures to yield a 3 S androgen receptor with uniform binding characteristics, and second, that the androgen receptors are distributed between two intra-nuclear pools--one containing about 10,000 molecules/nucleus sensitive to micrococcal nuclease digestion and salt and the other containing about 8,000 to 13,000 androgen receptors tightly bound to the nuclear matrix.
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PMID:Isolation of 3 S androgen receptors from salt-resistant fractions and nuclear matrices of prostatic nuclei after mild trypsin digestion. 686 57

In studies with a synthetic androgen, R 1881, an androgen-binding component was found in the cytosol of human placental villi. Kinetic analysis indicated that the Kd value of this component was 1.4 nM at 0-4 degrees C and that binding of R 1881 amounted to 277 +/- 73 fmol/mg protein. glycerol density gradient ultracentrifugation showed a peak of binding activity in the 8S region in a medium of low ionic strength, but in the 4.5S region in a medium containing 9.5 M KCl. The R 1881-binding component was inactivated by mild heat- or trypsin-treatment, but not by treatment with DNase or RNase. Most of the R 1881-binding activity was sedimented at 20 to 40% saturation of ammonium sulfate. These findings indicate that the R 1881-binding component in human placental cytosol is quite similar in its characteristics to androgen receptors, which are present in various androgen-responsive organs. Testosterone was a more potent competitor of R 1881-binding than DHT or cyproterone acetate. Scatchard plots indicated that the binding site of testosterone was identical with that of R 1881. These findings suggest that the androgen receptor in placental cytosol is specific for testosterone. The Kd value for testosterone was calculated to be 3.2 nM.
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PMID:Androgen receptor in human placental villi. 697 Dec 89

The relationship between the actions of androgen and thyroid hormone in induction of trypsin-like esteroproteases in mouse submandibular gland was studied. Zymograms prepared using tosyl-L-lysine alpha-naphthyl ester as substrate after isoelectric focusing in polyacrylamide slab gel showed that the isozymes of trypsin-like esteroprotease induced by 5 alpha-dihydrotestosterone and triiodothyronine were identical. The time courses of esteroprotease induction by these hormones were very similar, and the lag time was not shortened by treatment with both hormones. The doses of 5 alpha-dihydrotestosterone and triiodothyronine for half-maximal induction were 0.5 mg and 2.5 micrograms per 100 g body weight, respectively, and these values were not altered by simultaneous injection of other hormones. The binding capacity and affinity of androgen receptor for methyltrienolone, a synthetic androgen, were not affected by daily injections of triiodothyronine for 5 days. These results suggest that androgen and thyroid hormone act independently, not competitively, and so the two hormones induce trypsin-like esteroproteases additively.
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PMID:Independent inductions of trypsin-like esteroproteases by 5 alpha-dihydrotestosterone and triiodothyronine in mouse submandibular gland. 704 Mar 55


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