HPLC analysis of estrogen receptor by a multidimensional approach |
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| Authors: | N A Shahabi S M Hyder R D Wiehle J L Wittliff |
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| Affiliation: | 1. College of Environmental Science and Engineering, Beijing Forestry University, 35 Qinghua East Road, Haidian District, Beijing, 100083, PR China;2. Beijing Municipal Environmental Monitoring Center, Beijing, 100048, PR China;1. Bioanalytical Department, Synchron Research Services Pvt. Ltd., 5th Floor, the Chambers, Sarkhej-Gandhinagar Highway, Bodakdev, Ahmedabad 380054, India;2. Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380009, India;1. Southern Cross Plant Science, Southern Cross University, Lismore, NSW 2480, Australia;2. Yanco Agricultural Institute, NSW Department of Primary Industries, Yanco, NSW 2703, Australia;1. Plant Protection and Bimolecular Diagnosis Department, ALCRI, City of Scientific Research and Technological Applications, New Borg El Arab, Alexandria, 21934, Egypt;2. Forestry and Wood Technology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt;3. Plant Protection Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt;4. Facultad de Medicina Veterinaria Zootecnia, Universidad Autónoma del Estado de México, Estado de México, Instituto Literario, 50000 Toluca, México;5. Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt |
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| Abstract: | 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. |
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