Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.     

Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

Protein purification using a soluble affinity matrix: purification of estrogen receptor with estradiol-polylysine conjugate.

A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.     

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel

Abstract

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable.     

Partial purification of the 5-hydroxytryptamine-reuptake system from human blood platelets using a citalopram-derived affinity resin [corrected

This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific [3H]imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 microM citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after 125I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of [3H]imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and [3H]imipramine binding activity.     

Purification of skeletal muscle hexokinase by affinity elution chromatography

Type II hexokinase (EC 2.7.1.1) has been purified from rat skeletal muscle by a simple procedure involving chromatography on DEAE-cellulose, affinity elution chromatography from phosphocellulose, and gel filtration on Sephadex G-200. The key to the preparation of homogeneous enzyme is the affinity elution step in which an effector molecule, glucose 6-phosphate, is used as the eluting ligand. A 5300-fold purification is obtained by the procedure and over 400-fold purification is obtained in the affinity elution step alone. Approximately 3.3 mg of homogeneous hexokinase with a specific activity of 120 units/mg is obtained from 800 g of rat limb.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Murine macrophage tumors are a source of a 260,000-dalton acetyl-low density lipoprotein receptor

Subcutaneous injection of murine macrophage cell line P388D1 into syngeneic DBA/2 produced tumors, which upon solubilization with 40 mM octyl glucoside contained acetylated low density lipoprotein binding activity. The tumor-derived receptor specifically bound acetylated low density lipoprotein with an affinity of approximately 3 X 10(-8) M but did not bind low density lipoprotein or high density lipoprotein. It was identical in binding specificity, affinity, and Pronase sensitivity to the receptor in intact cells or that obtained from solubilized cultured cell membranes. Partial purification of the receptor was achieved by solubilizing tumors with 1% Triton X-100 followed by chromatography on polyethyleneimine cellulose. After elution with a NaCl gradient in the presence of octyl glucoside and association with liposomes, a 287-fold purification of the receptor was achieved. The receptor was identified by specific ligand blotting as a 260,000-dalton protein having a pI of approximately 6.0. Binding to the receptor by acetylated low density lipoprotein, malondialdehyde-modified low density lipoprotein, and maleic anhydride-modified serum albumin was demonstrated by ligand blotting. A single receptor protein can, therefore, account for the binding of multiple types of charge-modified lipoprotein and nonlipoprotein ligands to the macrophage cell surface.     

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.     

Purification of L-type pyruvate kinase from human liver by affinity chromatography on Blue-Dextran-Sepharose column

L-type pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40) was purified from human liver by an original method. This purification included toluene extraction, a-monium sulphate fractionation, DEAE-Sephadex bactchwise, CM-Sephadex batchwise with elective elution by ATP and affinity chromatography on a Blud Dextran-Sepharose column with specific elution by fructose 1, 6-diphosphate. This purification procedure allowed us to obtain 6 mg protein with a specific activity of 420 IU/mg protein, i.e. 2,690-fold purification with an overall yield of 34%. This preparation was homogeneous as judged by immuno-diffusion, acrylamide and sodium dodecyl sulphate acrylamide-gel electrophoresis.     

Visualization of the turkey erythrocyte β-adrenergic receptor

We have recently described the affinity chromatography purification of the turkey erythrocyte β-adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10-fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine-sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l-epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4–6 pmoles of ³H-dihydroalprenolol binding sites per mg of membrane protein. A single step of affinity chromatography on alprenolol-sepharose of the soluble digitonin extract resulted in an additional 1,000-fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations. Electrophoresis in SDS-polacrylamide of iodinated purified β-receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with β-mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l-propranolol. A fourth 52,000-dalton component (60,000 daltons after reduction with β-mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l-propranolol. Our results suggest that the β-adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.     

Partial purification of androgen receptor from hypertrophic human prostate

For purification of androgen receptor from hypertrophic human prostate, solutions used for elution of androgen receptor from DNA Sepharose, affinity labeling of the receptor and ability of affinity gel to retain the receptor were examined. Elution with 20 mM pyridoxal 5'-phosphate of the receptor from DNA Sepharose was more efficient than that with diluted pyridoxal 5'-phosphate, high ionic solution or various concentrations of Mg++, 3H-dihydrotestosterone bromoacetate was applicable to covalent binding with partially purified androgen receptor regardless of the low specificity of the ligand. Affinity gel of thiopropyl-Sepharose 6B coupled to 17 alpha-(2', 3'-epoxy-propyl)-5 alpha-dihydrotestosterone was better than Affigel 102 coupled to N-[3-(3-oxo-5 alpha-androstane-17 beta-yloxycarbonyl) propionyloxy] succimide or aminoethyl-Sepharose 4B coupled to 17 alpha-carboxyethynyl testosterone with respect to the rate of retention of androgen receptor. In view of these observations, the following purification procedures were constructed: Removal of DNA Sepharose-binders from the cytosol, 40% ammonium sulfate precipitation, affinity chromatography using thiopropyl-Sepharose 6B coupled to 17 alpha-(2',3'-epoxypropyl)-5 alpha-dihydrotestosterone, and DNA Sepharose chromatography. After affinity labeling of the receptor thus obtained, the molecular weight was estimated. Some 1300-fold purification with a yield of 0.25% of the androgen receptor was achieved. The molecular weight of the receptor was mainly 45 K with 90 K in a lesser amount. The Stokes radius was calculated as 30 A.     

Efficient two-step chromatographic purification of penicillin acylase from clarified Escherichia coli ultrasonic homogenate

A two-step chromatographic purification procedure from clarified Escherichia coli ultrasonic homogenate was evaluated. The capture step included immobilized metal affinity chromatography with Cu²⁺ as metal ion. Two elution methods were performed: 1 M NH₄Cl and 0.01 M imidazole. Respectively, we obtained a different purification fold (16.5 to 3.15) and a similar result for the recovery of activity (90–99%). The best elution method was chosen for the procedure. The second step, hydrophobic interaction chromatography, gave a 3.8-fold purification with 77.7% of activity. The total procedure gave a 66-fold purification in relation to the initial crude extract with 70% for the recovery of activity and was performed without any conditioning step and at the same pH value.     

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.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

A new method for the purification of RNA polymerase II (or B) from the lower eukaryote Physarum polycephalum. The presence of subforms

The DNA-dependent RNA polymerase II or B from the lower eukaryote Physarum polycephalum has been purified to apparent homogeneity by a new method employing poly(ethylene imine) precipitation and elution, and heparin-Sepharose affinity chromatography. The method is readily scaled up or down and affords a purification of over 5000-fold with a yield of 35-45%. The procedure is easy to perform and can be carried out in less than three days even on a large scale. Furthermore, it gives enzyme of higher purity and in at least 10-fold greater yield than previously published procedures for its purification from this organism. These improvements have allowed the detection of a series of subforms of the enzyme. The combination of precipitation using poly(ethylene imine) with chromatography on heparin-Sepharose may prove useful in the preparation of other proteins which interact with nucleic acids.     

Novel heterocyclic ligands for the thiophilic purification of antibodies

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.     

Purification of cellobiohydrolase I fromTrichoderma reesei using monoclonal antibodies in an immunomatrix

Summary The several components of the fungal cellulase system present practical problems in devising facile and efficient schemes for their purification. We report on a new single-step affinity chromatographic method for purification of cellobiohydrolase I ofTrichoderma reesei based on its selective absorption and elution using an immunomatrix constructed with CnBr-activated Sepharose 4B and monoclonal antibody specific for the enzyme. Isoenzymes of cellobiohydrolase I were purified directly from crude culture filtrate. The method is fast, simple, and of high resolution.     

The interaction of spermine with the ryanodine receptor from skeletal muscle.

The effect of polyamines on ryanodine binding activity of junctional sarcoplasmic reticulum membranes is described. Spermine stimulated the binding of ryanodine to its receptor up to 5-fold, with half-maximal stimulation obtained with 3.5 mM. Spermidine and putrescine also stimulated ryanodine binding, but they were about 12-fold less potent. The degree of stimulation is dependent on the NaCl concentration present in the assay medium. Spermine has no effect on the Ca(2+)-dependency of ryanodine binding but it increases the ryanodine binding affinity (Kd) by about 5.6-fold. Both the rate of ryanodine association with, and dissociation from, its binding site were affected by spermine. Spermine also stimulates the photoaffinity labelling by 3-O-(4-benzoyl)benzoyl[alpha-32P]ATP ([alpha-32P]BzATP) of the ryanodine receptor, increasing the BzATP binding affinity. We suggest that the stimulatory effect of spermine on ryanodine binding is due to its specific interaction with the ryanodine receptor. This spermine interaction enabled us to develop a new, one-step, fast and with high yield method for the purification of ryanodine receptor (Shoshan-Barmatz, V. and Zarka, A. (1992) Biochem. J. 284, in press).