Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

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.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Rat liver glucocorticoid receptor isolated by affinity chromatography is not a Mg2+- or Ca2+-dependent protein kinase

The glucocorticoid hormone receptor (92 kDa), purified 9000-fold from rat liver cytosol by steroid affinity chromatography and DEAE-Sephacel chromatography, was assayed for the presence of protein kinase activity by incubations with [gamma-32P]ATP and the photoaffinity label 8-azido-[gamma-32P]ATP. Control preparations isolated by affinity chromatography in the presence of excess steroid to prevent the receptor from binding to the affinity matrix were assayed for kinase activity in parallel. The receptor was not labeled by the photoaffinity label under photoactivation conditions in the presence of Ca2+ or Mg2+. A Mg2+-dependent protein kinase (48 kDa) that could be photoaffinity labeled with 8-azido-ATP copurified with the receptor. This kinase was also present in control preparations. The kinase could phosphorylate several minor contaminants present in the receptor preparation, including a protein (or proteins) of similar molecular weight to the receptor. The phosphorylation of 90-92-kDa proteins was independent of the state of transformation or steroid-binding activity of the receptor. These experiments provide direct evidence that neither the glucocorticoid receptor nor the 90-92-kDa non-steroid-binding protein associated with the molybdate-stabilized glucocorticoid receptor possesses intrinsic Ca2+- or Mg2+-dependent protein kinase activity.     

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.     

Purification of the Ah receptor from C57BL/6J mouse liver

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.     

Chick heat-shock protein of Mr = 90,000, free or released from progesterone receptor, is in a dimeric form

A monoclonal antibody (BF4) has been used to characterize and purify the heat-shock protein of Mr approximately 90,000 (hsp 90) present in the chick oviduct. In low salt cytosol, the sedimentation coefficient of hsp 90 is approximately 6.8 S, the Stokes radius approximately 7.1 nm, and the calculated Mr approximately 204,000, thus suggesting a dimeric structure. In 0.4 M KCl cytosol, only slightly smaller values were determined (approximately 6.5 S, approximately 6.8 nm, and approximately 187,000). Following purification by ion exchange and immunoaffinity chromatography, hsp 90 migrated as a single silver-stained band at Mr approximately 90,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient 6.2 S, the Stokes radius approximately 6.8 nm, and the Mr approximately 178,000 confirmed the dimeric structure. However, in both antigen or antibody excess conditions, only one molecule of monoclonal antibody could be bound to the hsp 90 dimer. Whether steric hindrance in a homodimer or the presence of two different 90-kDa proteins in a heterodimer explains this result cannot yet be decided. The dimer is not dissociated by high salt (1 M KCl) or the chaotropic agent (0.5 M NaSCN), but is disrupted by 4 M urea, suggesting a stabilization of the structure by hydrogen bonds. The molybdate-stabilized progesterone receptor hetero-oligomer form of approximately 8 S sedimentation coefficient was purified, and its hsp 90 component was then released by salt treatment. It was found to sediment at approximately 5.8 S and have a Stokes radius approximately 7.1 nm, giving Mr approximately 174,000. This observation is consistent with a previous report suggesting from specific activity determination, scanning of polyacrylamide gels, and cross-linking experiments that each purified nontransformed progesterone receptor molecule includes one progesterone binding unit per two 90-kDa protein molecules (Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., and Baulieu, E. E. (1984) Biochemistry 23, 6016-6023). This work brings direct evidence that both free hsp 90 and the non-hormone binding hsp 90 component released from the nontransformed steroid receptor in the cytosol are in a dimeric form.     

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.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Purification of unactivated progesterone receptor and identification of novel receptor-associated proteins

Progesterone receptor complexes were purified from crude cytosol in a rapid, gentle, one-step procedure using anti-receptor monoclonal antibodies covalently attached to an agarose resin. Five nonreceptor proteins specifically co-purified with unactivated avian progesterone receptor; these proteins had molecular masses of approximately 90, 70, 54, 50, and 23 kDa. The 90- and 70-kDa proteins have been previously identified as the 90-kDa heat shock protein and a member of the 70-kDa heat shock protein family, respectively. The 54-, 50-, and 23-kDa proteins have not been previously described as associated with avian progesterone receptor. Two-dimensional gel electrophoresis revealed charge heterogeneities for all five proteins. Except for p70, each could be dissociated from receptor by salt, a process inhibited by sodium molybdate. However, molybdate was not required for protein association with receptor in low ionic strength. Following progesterone treatment in vivo p70 still co-purified with cytosolic receptor although the other affiliated proteins were reduced, suggesting hormone-dependent dissociation in conjunction with receptor activation. One of the proteins, p54, displayed in vitro hormone-dependent dissociation which was not prevented by molybdate.     

Subunit composition of the molybdate-stabilized "8-9 S" nontransformed estradiol receptor purified from calf uterus

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.     

Interaction of chick oviduct progesterone receptor with immobilized aurintricarboxylic acid

Aurintricarboxylic acid (ATA) was immobilized on Sepharose 4B via a carbodiimide coupling mechanism. A majority of the chick oviduct progesterone receptor was retained on the affinity resin and could be recovered upon washing the column with buffer containing free ligand or 3 M guanidine-HCl. The [3H]progesterone-receptor complex retained its integrity following the chromatography on ATA-Sepharose as judged by sedimentation analysis. The procedure allowed significant purification of progesterone receptor: SDS-polyacrylamide gel electrophoresis of the purified preparation revealed elimination of many peptide bands present in the cytosol prior to ATA-Sepharose chromatography. The technique thus has a clear potential in characterization and purification of progesterone receptor.     

Purification of a heparin binding FGF receptor (HB-FGFR) from adult bovine brain membranes

A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies.     

Purification, characterization, and activation of the glucocorticoid-receptor complex from rat kidney cortex

The unactivated molybdate-stabilized glucocorticoid receptor (GcR) was purified from rat kidney cortex cytosol (RKcC) by using a modification of the procedure previously described by this laboratory for rat hepatic receptor. The purification includes affinity chromatography, gel filtration, and ion-exchange chromatography. The final preparation (approximately 1000-fold pure as determined from specific radioactivity) was used in subsequent physicochemical and functional analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single heavily Coomassie-stained band at 90 kilodaltons. Density gradient ultracentrifugation indicated a sedimentation coefficient of 10.5 +/- 0.05 S (n = 2). Chromatography on an analytical gel filtration column produced a Stokes radius (Rs) of 6.4 +/- 0.07 nm (n = 5). The Rs was unchanged when the molybdate-stabilized GcR was analyzed in the presence of 400 mM KCl or when analyzed in the unpurified (cytosolic) state. In contrast, the hepatic GcR was observed to exist as a larger form in cytosol (7.7 +/- 0.2 nm). Following purification, or upon gel filtration analysis under hypertonic conditions, the Rs was similar to that of the unpurified RKcC GcR. Following removal of molybdate from RKcC GcR and thermal activation (25 degrees C/30 min), DNA-cellulose binding increased 1.5-2-fold over the unheated control. Addition of RKcC or hepatic cytosol (endogenous receptors thermally denatured at 90 degrees C/30 min or presaturated with 10(-7) M radioinert ligand) during thermal activation increased DNA-cellulose binding an additional 2-6-fold beyond the heated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis induced in neuronal cells by C-terminal amyloid ß-fragments is correlated with their aggregation properties in phospholipid membranes

Rat brain proteins presenting high-affinity binding of S-adenosyl-L-homocysteine were solubilized and purified. Extraction of binding protein was carried out in the presence of Triton X-100 and 1 M NaCl; this solubilized fraction exhibits similar kinetic properties than the membrane proteins. Purification was performed using affinity chromatography on S-adenosyl-L-homocysteine carboxyhexyl Sepharose 48 conjugate. The analysis of the affinity gel eluate by SDS-PAGE showed high purification ratios for two proteins exhibiting 54 and 68 kDa. Three activity peaks were separated when solubilized membrane proteins were submitted to isoelectric focusing; the activity peaks corresponded to proteins of pH, 6.0, 6.5, and 7.2. SDS-PAGE separation of proteins contained in each peak showed protein aggregation; a 54-kDa subunit was present in each aggregate. Solubilized membrane proteins were labeled by photoaffinity labeling with tritiated S-adenosyl-L-homocysteine; the 54-and 68-kDa proteins were found among the specifically labeled proteins. Finally, according to the previous data from the literature, the purified S-adenosyl-L-homocysteine binding proteins do not seem to be the same as adenosine receptors or phosphatidylethanolamine-N-methyltransferase.     

Rapid purification of protein kinase C from rat brain. A novel method employing protamine-agarose affinity column chromatography

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine-coupled to agarose. Sequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and protamine-agarose columns resulted in a 1,500-fold purification of protein kinase C. SDS-PAGE analysis of the purified enzyme resolved a doublet protein of 77-80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments. The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column; phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.     

Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli

Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100. Affinity chromatography using an amylose column and Ni2+ affinity column was used to purify the sigma-1 receptor. The sigma-1 receptor purified by this method is a 26 kDa polypeptide as assessed by SDS-PAGE, binds sigma ligands with high affinity and can be specifically photoaffinity labeled with the sigma-1 receptor photoprobe, [125I]-iodoazidococaine. Ligand binding using [3H]-(+)-pentazocine indicated that approximately half of the purified protein in Triton X-100 bound to radioligand. The MBP-sigma-1 receptor and the sigma-1 receptor in 0.5% triton were maximally stable for approximately two weeks at -20 degrees C in buffer containing 30% glycerol.     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.     

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.