Abstract
Limited trypsin digestion of the estradiol-receptor complex in human mammary cancer cytosol changed the sedimentation coefficient of the complex from 8 to 3–4 S. Gel filtration in high ionic strength on Sephadex columns calibrated with reference proteins showed that, after trypsinization, the Stokes radius of the estradiol-receptor complex decreased from 2.8 to 1.9 nm. Isoelectric focusing analysis indicated that the 8 S form of the steroid-receptor complex precipitated during electrofocusing in glass columns, whereas the trypsinized receptor focused as a sharp peak at pH 6.6. Based on these findings a method was developed for isoelectric focusing of the partially trypsin-digested estradiol-receptor complex in human mammary cancer cytosol on slabs of polyacrylamide gel. With this technique, the focusing time is 2 hr, at least eight different tumors may be analyzed on one gel, and sample volumes up to 300 μl are easily applied. Furthermore, electrofocusing of the estradiol-receptor complex was compared to sucrose gradient centrifugation analysis in low ionic strength and was found to be more sensitive and more specific. We therefore suggest that the described technique of isoelectric focusing should be considered as an alternative to sucrose gradient centrifugation for estradiol receptor assays.
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