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T Ratajczak

Publications and source records attributed to T Ratajczak.

At least 37 records · Page 2Linked to original sources
PubMed1995

Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli.

Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C. The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein.

Amino Acid Isomerases↗
PubMed1993

The cyclophilin component of the unactivated estrogen receptor contains a tetratricopeptide repeat domain and shares identity with p59 (FKBP59).

Using a rapid single-step affinity chromatography procedure we have isolated the unactivated estrogen receptor from bovine uterus. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analyses for protein extracts recovered from affinity chromatography of receptor cytosols, either preincubated or untreated with estradiol, suggest a component structure for the intact oligomeric receptor which includes hsp90, hsp70, p59, a 40-kDa cyclophilin-related protein, and an uncharacterized 22-kDa protein species. We have chemically determined the amino acid sequences of eight peptides derived from the 40-kDa component and now report the cloning and primary sequence of a cDNA encoding this protein, which is designated estrogen receptor-binding cyclophilin (ERBC). Homology analyses confirm that ERBC is a new member of the cyclophilin family and contains a C-terminal domain with significant sequence homology to an internal region of p59, a binding protein for the immunosuppressant FK506 (FKBP59). This conserved region includes a 3-unit tetratricopeptide repeat domain bounded at the C terminus by a putative calmodulin binding site. We propose that the tetratricopeptide repeat domain mediates the protein interaction properties of ERBC and p59. Both immunophilins may have important roles in receptor assembly and may represent a new category of ligand- and calcium-dependent modulators of protein function.

Amino Acid Isomerases↗
PubMed1992

Multiple elements within the glucocorticoid regulatory unit of the rat alpha 1-acid glycoprotein gene are recognition sites for C/EBP.

Sequences between -106 and -42, located immediately downstream of the glucocorticoid response element, are essential for efficient glucocorticoid-stimulated expression of the alpha 1-acid glycoprotein (AGP) gene. We have used mobility shift assays with oligonucleotides bearing wild type and mutated sequences from segmented portions of this region to characterize the specific interaction of similar binding factors from rat liver and HTC rat hepatoma cell nuclear extracts. One of these factors, AGP nuclear factor 2 (ANF-2), appears capable of dual interaction with the homologous recognition sites, HA (-133 to -104) and HB (-81 to -72), which overlap and are located downstream of the glucocorticoid response element, respectively. Using an affinity matrix containing the HB sequence we have isolated ANF-2 from rat liver nuclear extracts. On the basis of immunological evidence rat liver ANF-2 was confirmed to be highly related and probably identical to CCAAT/enhancer-binding protein (C/EBP). Methylation protection analyses with partially purified, rat liver ANF-2 confirmed HA and HB as recognition sites for C/EBP-related factors and are consistent with the location of a third interaction site for these transactivating proteins at HX (-102 to -93). We propose that the sequences HA, HX, and HB, spanning residues -113 to -72 of the AGP promoter, might serve as recognition sites for a family of C/EBP-like nuclear factors that coordinate the glucocorticoid-mediated induction of the AGP gene.

Animals↗
PubMed1991

AGP/EBP(LAP) expressed in rat hepatoma cells interacts with multiple promoter sites and is necessary for maximal glucocorticoid induction of the rat alpha-1 acid glycoprotein gene.

Transcription of the rat alpha 1-acid glycoprotein (AGP) gene is induced by glucocorticoids. In addition to the glucocorticoid response element which maps to bases -120 to -107, sequences located between bases -106 to -42 have been shown to be necessary for hormone induction. We have previously identified multiple sites of C/EBP interaction with the AGP promoter in the region -106 to -64. In this study, we purify and identify a C/EBP family member, AGP/EBP(LAP), present in the rat hepatoma cell line HTC (JZ.1) which also binds to the C/EBP recognition sites in this region. Mutations in the recognition sites that prevent binding are analyzed, and the results suggest a positive as well as a possible inhibitory role for AGP/EBP(LAP) in the glucocorticoid induction of the gene in HTC (JZ.1) cells.

Animals↗
PubMed1990

Isolation of untransformed bovine estrogen receptor without molybdate stabilization.

A low concentration estrogen-derivatized affinity resin has been used in a rapid, single step purification of the untransformed estrogen receptor from calf uterine cytosols prepared without sodium molybdata. The procedure isolates the Mr 65,000 estrogen receptor in association with the bovine heat shock protein hsp90. Small amounts of proteolyzed receptor ranging in size from Mr 50,000 to 60,000 are also present in the purified extracts. Results from affinity chromatography of receptor cytosols either untreated or presaturated with estradiol suggest that two proteins of Mr 22,000 and 38,000 are co-purified with the untransformed receptor complex and may represent additional nonhormone-binding components of the native receptor form. Some indication of the stability of protein-protein interactions within the oligomeric complex has been derived from differential salt elution studies with heparin-sepharose and affinity gel-immobilized untransformed receptor. On size exclusion high performance liquid chromatography the untransformed complex eluted with a Stokes radius of 75 +/- 2 A (n = 18), but was shown to be sensitive to extended ultracentrifugal analysis dissociating to the receptor homodimer, sedimentation coefficient 5.3 +/- 0.3 s (n = 5). Preliminary data on urea- and heat-induced transformation of the isolated receptor to the DNA-binding state is presented.

Animals↗
PubMed1990

Interaction of bovine estrogen receptor with immobilized zinc.

The metal-binding properties of partially purified untransformed or salt-dissociated bovine estrogen receptors were studied using zinc-chelated iminodiacetic acid gels. Only the salt-dissociated 5S receptor is retained by the metal-chelated resin, and this interaction is dependent on the presence of dithiothreitol. The untransformed 9S receptor is not retained, indicating that the zinc-interacting amino acid residues may be masked by receptor-associated proteins such as 90K heat-shock protein or because of an unfavorable receptor conformation.

Animals↗
PubMed1989

The interaction site for tamoxifen aziridine with the bovine estrogen receptor.

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Strömstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and Jörnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.

Amino Acid Sequence↗
PubMed1988

Chemical characterization by protein sequence analysis of the bovine estrogen receptor.

Tryptic peptides generated from bovine estrogen receptor have been fractionated and purified using microbore column high performance liquid chromatography. Sequence analysis performed on six of these peptides, derived from diverse structural regions of the receptor protein, yielded 73 unique assignments corresponding to approximately 12% of the molecule. The amino acid sequences of these peptides displayed a high degree of similarity with corresponding sequences from estrogen receptors of mammalian origin, but were only moderately conserved in receptors from non-mammalian species. The sequenced residues of one tryptic peptide, positioned in the estrogen binding domain, were fully conserved in all estrogen receptors.

Amino Acid Sequence↗
PubMed1988

Sequence analysis of the nonsteroid binding component of the calf uterine estrogen receptor.

Microbore reversed-phase high performance liquid chromatography has been utilized to fractionate and purify a number of tryptic peptides generated from the 90K nonsteroid binding component of the calf uterine estrogen receptor. Sequence analysis was performed on six peptides yielding 78 unique amino acid assignments, this corresponds to approximately 10% of the molecule. These peptides share sequence similarities with three heat shock proteins, Drosophila hsp 83 (83% homologous), yeast hsp 90 (55%) and chicken hsp 108 (32%). The amino acid composition of the protein indicates a prevalence of charged amino acid residues.

Amino Acid Sequence↗
PubMed1988

Structural characterization of the 9-10S estradiol receptor from calf uterus.

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.

Animals↗
PubMed1987

The amino-terminal sequence of the 85-90K nonhormone binding component of the molybdate-stabilized estradiol receptor from calf uterus.

The first six N-terminal amino acid residues of the 85-90K non-estrogen binding component of the calf uterine, molybdate-stabilized estradiol receptor have been determined by Edman degradation. After affinity chromatography of the stabilized receptor oligomer, the 85-90K unit was purified to homogeneity by preparative gel electrophoresis using electroelution for protein recovery. Inverse-gradient high performance liquid chromatography provided the 85-90K protein suitable for amino-terminal sequence analysis.

Amino Acid Sequence↗
PubMed1985

Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus.

The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol.

Animals↗
PubMed1983

Purification to homogeneity of the nuclear estrogen receptor from chick liver.

The nuclear estradiol receptor from chick liver was purified to apparent homogeneity by using a combination of ammonium sulfate precipitation and affinity gel chromatography. The purified receptor migrated as a single band on an SDS-polyacrylamide gel with a molecular weight of 55000 and it exhibited a sedimentation coefficient of 4 S, a dissociation constant of 1.13 nM and a steroid specificity resembling that of the receptor in crude extracts.

Animals↗
PubMed1982

Requirement for a reduced sulphydryl entity in the protection of molybdate-stabilized estrogen receptor.

The separate and combined effect of molybdate and dithiothreitol on the stability of human uterine 9 S estrogen receptor were studied. Maximal, short-term, protection of the 9 S estrogen receptor was achieved by the joint inclusion of both stabilizing agents in cytosol buffers. This molybdate--dithiothreitol-mediated stability was dependent on reducing agent concentration inferring sulphydryl involvement in 9 S receptor protection by molybdate. The study also showed that molybdate--dithiothreitol could not prevent the gradual decay of the 9 S estrogen receptor to the 4 S form in cytosols stored at 4 degrees C over prolonged periods.

Buffers↗
PubMed1981

Determination of SHBG-bound sex hormones by selective ammonium sulphate precipitation.

This paper describes a direct method for determining sex hormone binding globulin (SHBG)-bound sex hormones in human plasma after separation of SHBG-bound and unbound hormone fractions by selective precipitation with ammonium sulphate. In normal women variations in SHBG-bound and -free hormone generally paralleled fluctuations in total hormone. Changes in SHBG-free estradiol did not have any marked effect on plasma SHBG and sHBG-free testosterone. Our results suggest a buffer role for SHBG through which the biological response to sudden changes in sex hormone concentration is moderated.

Adult↗
PubMed1981

The synthesis and study of some potential affinity labeling reagents for estrogen receptors.

The influence of the following affinity labeling reagents on the binding of tritiated estradiol-17 beta (E) by human and calf uterine cytosols was studied: 11 beta-chloromethylestradiol (ORG4333), 2-azidoestradiol (2A-E), 4-azidoestradiol (4A-E), 3-azidohexestrol (3A-H), estradiol-17 beta 17-bromoacetate (E-17BrAc), 6-[O-carbo-(2'-chloroethoxy)methyl] oximinoestradiol (6-CMOEtC1), 17-[O-carbo-(2'-chloroethoxy) methyl] oximinoestrone (17-CMOEtCl), 2-di (2'-hydroxy-3'-chloropropyl)aminoestradiol (E-Mustard). For the human uterine estrogen receptor the relative binding affinity decreased in the order E greater than ORG 4333 greater than E-17BrAc greater than 3A-H greater than 2A-E greater than 4A-E greater than 6-CMOEtCl greater than E-Mustard greater than 17-CMOEtCl. The binding characteristics of the calf uterine estrogen receptor were qualitatively similar, but quantitatively different. ORG 4333 appeared to form a highly stable association with the receptors, but alkylation of the protein could not be conclusively demonstrated.

Affinity Labels↗