AN IMPROVED METHOD FOR THE QUANTIFICATION AND RECOVERY OF RAT UTERINENUCLEAR TYPE-II [H-3] ESTRADIOL BINDING-SITES IMMOBILIZED ON A GLASS-FIBER MATRIX

An improved assay for measuring ligand binding to extracted nuclear ty pe II estrogen binding sites which involves preimmobilization on glass fiber filters is described. At least two classes of specific estrogen binding sites have been demonstrated in rat uterus as well as in a va riety of other tissues and species and have been designated as type I and type II. Although the endogenous ligand to the type II binding sit e has recently been identified as methyl p-hydroxyphenyllactate (MeHPL A), tritiated estrogens are generally used for radiolabeling this site due to the susceptibility of MeHPLA to enzymatic hydrolysis In In vit ro assays. After extracting the type II site from the nuclear matrix, ligand binding and protein stability appear to be significantly enhanc ed by first immobilizing the site on an artificial matrix, such as hyd roxylapatite, before incubating with radiolabeled ligand. Immobilizati on of the extracted site on glass fiber filters results in higher spec ific binding and lower nonspecific binding when compared to hydroxylap atite and a number of other immobilization matrices. The glass fiber l igand exchange procedure for measuring type II binding can also be per formed on smaller samples and requires less time than other methods. T ype II sites are significantly stabilized when immobilized on glass an d exhibit sigmoidal binding curves when incubated with increasing conc entrations of [H-3]estradiol and [H-3]estrone and display inhibition d ata characteristic of that observed using more traditional assays. Sin ce the type II binding site can be readily extracted from glass, glass immobilization provides a very rapid method for the desalting and con centration of type II sites prior to subsequent characterization and p urification steps and has implications for the electroblotting and pos tlabeling of type II preparations purified using nondenaturing electro phoresis.