Salman M. Hyder

High-performance hydrophobic-interaction chromatography of steroid hormone receptors.

The use of high-performance hydrophobic-interaction chromatography (HPHIC) on SynChropak 500 propyl columns has been evaluated for the first time in the analysis of estrogen receptors labeled with [125I]iodoestradiol-17 beta. These receptors were extracted from reproductive tissues with 500 mM phosphate buffer and applied to the stationary phase. Utilizing an inverse phosphate gradient (500 to 10 mM), elution resulted in rapidly excluded components in the void volume followed by a second radioactive peak at 400 mM phosphate. Both peaks appeared to contain specific estrogen-binding components in that steroid association was inhibited by diethylstilbestrol and free ligand was eluted with a different retention time. A great deal of [125I]iodoestradiol-17 beta was retained by the column. Inclusion of acetonitrile (20%) in the mobile phase resulted in the elution of [125I]iodoestradiol-receptor complexes at a different position from free ligand. Distribution of specific estrogen-binding components appeared to be tumor-dependent. These preliminary results indicate that HPHIC may be useful for isolating various isoforms of steroid hormone receptors so that detailed information regarding their intrinsic properties may be ascertained.

Characterization of estrogen receptors and associated protein kinase activity by high-performance hydrophobic-interaction chromatography

We have determined that high-performance hydrophobic-interaction chromatography (HPHIC) with weakly hydrophobic columns permit the rapid separation of the labile isoforms of estrogen receptor proteins. Previously we reported the use of the SynChrom propyl 500 column for HPHIC of steroid receptors. However, due to the strongly hydrophobic characteristics of the ligand, [125I]iodoestradiol-17 beta, and the receptor protein, organic solvent was required in the mobile phase for greater recovery of receptor proteins. Here, we report separation of steroid receptors from human breast tumors and rat uteri, using the Beckman CAA-HIC, a non-ionic polyether-bonded column, without the need for organic solvents and with virtually 100% recoveries. Receptors were extracted in 10 mM phosphate buffer (pH 7.4). Maximum resolution and separation were achieved when a descending salt gradient of ammonium sulfate in phosphate buffer (pH 7.4) was used (2-0 M in 30 min). Estrogen receptor (ER) was resolved into two isoforms with tR = 22 +/- 1 min (n = 16, designated as peak I) and 27.5 +/- 0.5 min (n = 14, designated as peak II) and a purification of five- to twenty-fold in a single pass. Free steroid was eluted at tR = 35 +/- 1 min (n = 4). Separation was dependent on adjusting the ionic strength of cytosol to 1.5 M ammonium sulfate. ER, purified by HPHIC, retained ligand binding capacity and exhibited protein kinase activity, which was dominant in the less hydrophobic peak I (tR = 22 min) when immunoprecipitated with the monoclonal antibody D547. This method of rapidly purifying ER with high retention of biological activity may now be applied to the study of the molecular interrelationships of steroid receptor isoforms.

Interaction of estrogen receptor isoforms with immobilized monoclonal antibodies

High-performance liquid chromatography was performed to separate the various isoforms of estrogen receptor from human breast cancer, based on size (high-performance size-exclusion chromatography) and surface charge (high-performance ion-exchange chromatography) properties. The ability of these isoforms to interact with the monoclonal antibodies was assessed. All isoforms exhibited similar immunodeterminant sites, but when they are bound to [125I]iodoestradiol-17 beta (IE), only 30% binding of the radioactive complex to the immobilized monoclonal antibodies was observed. However, the mass of the receptor recognized by the antibody bead, via the estrogen receptor-enzyme immunoassay (ER-EIA), was always significantly higher. This was true for both fractionated and non-fractionated cytosols, suggesting that non-ligand binding forms, such as precursors and products of the estrogen receptor, were also recognized; or the ligand was only selecting for a particular conformer(s); or the monoclonal antibody on the bead recognized other proteins associated with estrogen receptor. Ion-exchange fractionation of unlabeled receptor showed loss of immunodeterminant sites. However, size-exclusion fractionation did not show this effect. Diethylstilbestrol, a competitor of IE binding, showed marked stability of receptor recognized by ER-EIA during both size-exclusion and ion-exchange chromatography. Limited trypsin treatment of the receptor caused the loss of immunodeterminant sites without altering the ligand binding sites. Thus, proteolysis of estrogen receptors in cytosols of human breast cancer could easily lead to underestimation by ER-EIA. Although the components with immunodeterminant sites recognized by ER-EIA were always eluted with the ligand-binding isoforms of the estrogen receptor, our data suggest that the concentration of the protein having the epitope associated with the monoclonal antibody is unequal to that recognized by the steroid ligand. We conclude that application of ER-EIA to clinical assays of estrogen receptors clearly needs further clarification.

HPLC analysis of estrogen receptor by a multidimensional approach

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 A isoform and by one appearing after the V0 (approximately 60 A) in human uterus. However, in endometrial carcinoma other smaller binding components with Stokes radii of less than 20 A were observed also. In buffers containing 400 mM KCl, predominantly a 28-30 A species was observed by HPSEC. Further characterization of the 28-30 A isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8-9 min), and the other at 50-70 mM phosphate. The elution profile of the larger species (approximately 60 A by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50-70 mM phosphate and a major isoform at 180 mM phosphate. However delayed analysis (after 2 h) of the 60 A isoform showed a similar profile (components in buffer wash and at 50-70 mM phosphate) obtained with the 30 A species. This time dependent change was not observed for the 30 A species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28-30 A and 60 A species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.

High-performance hydrophobic interaction chromatography of estrogen receptors and magnesium dependent protein kinase(S): Detection of two molecular forms of estrogen receptors in the presence and absence of sodium molybdate

The separation characteristics of estrogen receptors (ER) from human breast cancer were evaluated based on their hydrophobic properties. Results show that (1) two distinct hydrophobic isoforms of ER exist either in the presence of sodium molybdate (peaks MI and MII with retention times of 15-17 min and 24-26 min) or in its absence (peaks I and II with retention times of 25-27 min and 34-36 min respectively); (2) this is observed whether molybdate (MoO2-4) is added to prepared cytosol or to the buffer prior to homogenization; (3) isoform MII and I separated with similar retention times suggesting they are the same ER species; and (4) isoform MI (Rt = 15-17 min) is a distinct ER species from either MII/I (Rt = 25-28 min) or II (Rt = 34-36 min). The latter isoform represents a highly hydrophobic species seen only in the absence of MoO2-4. Finally, (5) MoO2-4 ions appear to interconvert the most hydrophobic species (II) into the least hydrophobic isoform (MI) with virtually no change in the quantity of isoform(s) MII/I. However, it cannot be ascertained if the II----MI interconversion proceeds via isoform MII/I. Isoform II may result from the interaction with the stationary phase via its DNA binding site since MoO2-4, which is suggested to directly interact with this site, selectively interacts with peak II. These results imply the usefulness of inclusion of receptor stabilizing reagents in the mobile phase for preserving receptor integrity and in elucidating the interrelationships of ER isoforms and associated macromolecules.

Detection of two high molecular weight hydrophobic forms of the human estrogen receptor

The human estrogen receptor gene encodes a single protein of molecular weight 65,000 daltons. However, using a sensitive and rapid technique of high-performance hydrophobic interaction chromatography we have detected two distinct estrogen receptor species both of which are high molecular weight proteins (ca. 60A) as determined by high-performance size-exclusion chromatography. These are detected either in the presence or absence of sodium molybdate; rechromatography of individual isoform indicates that the two protein complexes have independent hydrophobic contact points. Consistent elution patterns of the two receptor species indicates they are formed selectively. We conclude that different post-translational modifications of the estrogen receptor protein could allow their specific interaction with non-receptor components resulting in the formation of two distinct high molecular weight complexes which would be rapidly resolved by high-performance hydrophobic interaction chromatography.

Rapid purification of topoisomerase I from human breast cancer cells by high-performance liquid chromatography

The DNA regulatory enzyme topoisomerase I (TpI) from human breast cancer cells has been analyzed by high-performance liquid chromatography (HPLC) for the first time. Cells were homogenized in Tris buffer and TpI activity was extracted with 0.5 M sodium chloride. Negatively supercoiled plasmid pBR322 was used as the substrate to monitor TpI activity, as judged by relaxed products, analyzed on 1% agarose gels. HPLC in the anion-exchange mode (HPIEC) provided an approximately 6-fold purification of the enzyme. Enhanced purification was subsequently obtained by chromatography of a HPIEC eluate on size-exclusion columns (30- to 60-fold). Recovery of TpI from size-exclusion columns, whether used in multistep analysis or as the first step, was dependent on inclusion of organic solvent, 1-propanol (0.5%, v/v), in the mobile phase. Marked resolution of TpI activity was observed with HPIEC on a SynChrom CM-300 column. Enzyme activity was noted in the void volume, at 150-200 mM phosphate and at 250-350 mM phosphate. TpI purification was 10- and 120-fold in the latter two peaks, respectively. Silver-stained polyacrylamide gels of TpI-containing activity, eluted from a CM-300 column, showed considerable purification of all but the void volume fraction. A distinct protein band at approximately 88-90 kD was seen in the peak eluted from the CM-300 column with 250-350 mM phosphate. These results indicate that HPLC is useful for rapid purification of the labile enzyme, TpI, in the analysis of its structure-function relationship.

Separation of two molecular forms of human estrogen receptor by hydrophobic interaction chromatography : Gradient optimization and tissue comparison

High-performance hydrophobic interaction chromatography (HPHIC) was used to separate and characterize two molecular forms of estrogen receptor with a SynChropak propyl hydrophobic column (300 A pore size). The linear gradient utilized earlier with a polyether-bonded column (2 to 0 M) ammonium sulfate in 40 min, gave poor resolution with the propyl column. However, resolution was maximized with either an initial ammonium sulfate concentration of 1 M (40-min gradient) or with a two-phase gradient (2 to 0.5 M in 10 min, 0.5 to 0 M in 30 min). This indicated that the propyl column was more hydrophobic than the polyether column. Estrogen receptor separated into two isoforms, either in the presence [MI, retention time (tR) = 13-14 min; MII, tR = 20-21 min] or absence (I, tR = 21-23 min; II, tR = 31-33 min) of the estrogen receptor stabilizing reagent, sodium molybdate. Similar isoforms were observed in cytosols from human breast tumors, uterus, and MCF-7 breast cancer cells. Unlike others, MCF-7 estrogen receptor did not show MI. Since MCF-7 cells contain 90,000 dalton heat shock proteins (HSP-90), HSP-90 is probably not directly involved in MI formation. Sodium molybdate selectively interacted with isoform II and converted it to MI. All isoforms appeared to be high-molecular-weight proteins (greater than 60 A) when subsequently analyzed by high-performance size-exclusion chromatography. Interestingly, when estrogen receptor was immobilized on the stationary phase, no change was detected in either hydrophobicity or steroid-binding capacity. After 16-18 h, immobilized receptor was eluted with a slightly longer tR. During incubation on the column, component MI was converted into I and/or II. HPHIC appears to be a rapid, yet gentle procedure for isolating large receptor complexes in significant quantities with high recoveries. This allows one to discern the complicated structure-function relationships of estrogen receptor and associated non-receptor proteins and provides information about the on-column behavior of complex proteins.

High-performance hydrophobic interaction chromatography as a means of identifying estrogen receptors expressing different binding domains

Methodology for high-performance hydrophobic interaction chromatography (HPHIC) of estrogen receptors (ER) was developed, utilizing a polyether-bonded stationary phase, which was non-ionic in nature. Using a descending salt gradient (2 M to 0 M ammonium sulphate in 40 min), ERs from human breast cancer separated into two isoforms, which retained ligand-binding domains. The same isoforms were observed with ER preparations from rat uterus. When sodium molybdate, a stabilizer of receptor structure, was incorporated into the mobile phase, it altered the ER characteristics, producing an earlier elution of one component, while the other one remained unchanged. Treatment of breast cancer cytosol with RNase A did not alter ER elution from either the hydrophobic or size-exclusion (TSK 3000 SW) columns. Modification of cysteine residues with N-ethylmaleimide led to a broad elution pattern of receptor from the hydrophobic column, implying the existence of multiple conformations of ER. Limited trypsin treatment of ER, which removes the DNA binding domain, led to the elution of only one receptor peak from the hydrophobic column. The receptor eluted at 24 min both in the presence and in the absence of sodium molybdate. Thus, at least one mechanism of the sodium molybdate effect must involve its direct interaction with ER to influence the sequence between the DNA-binding domain and the N-terminus. This also indicates that the most hydrophobic species of ER (sodium molybdate sensitive) may arise due to the interaction of the DNA-binding site with the stationary phase. Other possibilities, such as differential post-translational modifications of the receptor protein could also account for the two isoforms of ER, observed in HPHIC analysis.

Structural Features and Clinical Significance of Estrogen Receptors

An important question in molecular endocrinology is how structurally simple molecules, such as the steroid hormones, initiate the myriad of effects in a wide variety of target organs via their receptor proteins. It is generally accepted that a prerequisite for responsiveness to a steroid hormone stimulus is a cellular protein termed the steroid hormone receptor or steroid binding protein. These receptor proteins have been found in concentrations ranging from 50–50,000 sites per target cell but are virtually absent in nontarget cells. A biologically important property is the association of a steroid hormone with its characteristic receptor protein in a manner exhibiting both high affinity and ligand specificity.