Abstract
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.
Highlights
We describe an affinity chromatography procedure for the purification of the molybdate-protected 9-10 S estradiol receptor from calf uterus
Receptor Purification-The 9-10 S estradiol receptor from calf uterus was purified by sequential chromatography on heparin-Sepharose and anestrogen-linked affinity resin
During high salt-mediated elution of the receptor from heparin-Sepharose, a high degree of protection against dissociation of the large receptor form was obtained with buffer containing molybdate (PMD buffer)
Summary
Receptor Purification-The 9-10 S estradiol receptor from calf uterus was purified by sequential chromatography on heparin-Sepharose and anestrogen-linked affinity resin. High molecular weight receptor forms were observed on low salt ultracentrifugal analysis of calf uterine cytosols prepared in PD buffer, with or without molybdate (Fig. 2,A and C). During high salt-mediated elution of the receptor from heparin-Sepharose, a high degree of protection against dissociation of the large receptor form was obtained with buffer containing molybdate (PMD buffer). In the presence of0.4 M KC1, the predominant complex sedimented at 8.3 f 0.2 S (n= 5) (Fig. 2B). the sedimentation profile suggested a mixture of binding forms, analytical size-exclusion HPLC’ in hypotonic PMD buffer revealed only one sharp and symmetrical peak of radioactivity (not shown) corresponding to that observed for cytosolic receptors (& = 74 -+ 1 8, (n = 5)). Full size photocopies are included in the microfilm edition of the Journal thaist available from Waverly Press.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
