Biomedical subjects

S M Hyder

Publications and source records attributed to S M Hyder.

43 records · Page 3Linked to original sources

PubMed1988

Alterations in estrogen receptor isoforms in the mammary gland and uterus of the rat during differentiation.

1. Estrogen receptors in lactating mammary glands and uteri of rats which were 10 and 19 days postpartum exhibited molecular heterogeneity based on their surface charge properties. 2. The polymorphism of estrogen receptors detected by high-performance ion exchange chromatography may be monitored in-line with a radioisotope detector. 3. Estrogen receptors from the mammary gland and uterus of rats at 10 days of lactation exhibited primarily two receptor isoforms eluting at 200-250 mM and 250-300 mM phosphate, whereas three ionic isoforms (eluting at 50-150, 200-250 and 325-375 mM phosphate) were found in the mammary glands of rats at 19 days of lactation. Similar changes in the profiles of estrogen receptor isoforms were observed in uterine cytosol preparations at each stage of postpartum differentiation. 4. The elution pattern of receptor-associated radioactivity was not altered by the addition of diisopropylphosphate, a potent inhibitor of trypsin-like proteases, either before, during or immediately after homogenization. This indicates that the differences observed in the receptor elution profile of 10 and 19 day postpartum lactating mammary glands were not due to artifactual proteolysis. 5. In summary, our data indicate that the differentiation stage of lactating mammary glands may dictate the final profile of receptor isoforms detected.

Animals↗

PubMed1987

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.

Ammonium Sulfate↗

PubMed1987

Progestin receptors from tissues either exhibiting or lacking estrogen response mechanisms. Comparison of conventional and high-performance liquid chromatography methodology.

Evidence from a variety of target organs has shown that progesterone receptor (PR) is induced by estrogen receptor (ER) in normal and neoplastic tissues. However, approximately 12% of the normal human uterine samples exhibit only PR with no measurable ER, suggesting the expression of both inducible and constitutive receptor isoforms. We investigated several molecular properties of PR from tissues either exhibiting or lacking ER. All studies were conducted in potassium phosphate buffer containing 10 mM sodium molybdate with a synthetic progestin, [3H]R5020 as the ligand. Radioinert R5020 was used as competitor to assess nonspecific association. Competition analysis showed that PR from both sources exhibited similar ligand specificities and affinities. Relative affinities were ORG 2058 greater than R5020 greater than medroxy-progesterone acetate greater than progesterone much greater than testosterone (Kd values ranged from 10(-9) to 10(-10) M; testosterone showed no specific competition). We utilized high-performance liquid chromatography in the size-exclusion (HPSEC) and ion-exchange (HPIEC) modes to probe the size and ionic properties of PR. HPSEC profiles showed that the PR isoform from both sources was eluted as a single, sharp peak greater than 75 A. HPIEC elution profiles indicated no differences in the surface ionic properties in that PR from both tissue types eluted with ca. 100 mM phosphate. These experiments show no difference between the inducible and the putative constitutive form of PR. Thus, some PR species may not require estrogen for their formation.

Binding, Competitive↗

PubMed1986

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.

Breast Neoplasms↗

PubMed1986

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.

Antibodies, Monoclonal↗

PubMed1986

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 Stoke's 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.

Centrifugation, Density Gradient↗

PubMed1985

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.

Breast Neoplasms↗