Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing.

We describe the purification of a single-strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative-scale isoelectric focusing. Protein separation takes place entirely in solution, with shear-stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE-Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially-purified protein (250 mg, 6700 U/mg) was focused in pH 3-4 carrier ampholytes. 67% of the activity was recovered in pooled peak fractions with a specific activity of 54,000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four-fold increase in specific activity (from 4000 U/mg protein to 15,000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.

Characterization of rabbit uterine estrogen receptor proteins by radioiodination and partial peptide mapping.

Two estrogen binding proteins (Mr = 50,000 and 65,000) were purified from rabbit uterine cytosol using an improved procedure for affinity chromatography on diethylstilbestrol-agarose. The estrogen receptors were radioiodinated while adsorbed to the resin using the lactoperoxidase or Bolton-Hunter techniques. After elution, the labeled receptors were utilized for peptide mapping studies and investigations of receptor function. Partial peptide mapping revealed strong homology between the Mr 50,000 and 65,000 proteins suggesting common structural features. Estrogen receptors labeled by the lactoperoxidase procedure were rendered unable to bind immobilized heparin or hormone; in contrast, the Bolton-Hunter labeling technique yields proteins that retain both their ability to bind hormone and to absorb on heparin-agarose. The development of these iodination methodologies appears useful for the investigation of both the structure and functional properties of the receptor proteins.

An immobilized antiestrogen binds a specific uterine protein in addition to estrogen receptor proteins.

An antiestrogen affinity resin was synthesized by conjugating LY117018, a benzothiophene -derived antiestrogen, to epoxy-activated agarose. This affinity resin bound the Mr = 50 000 and 65 000 estrogen receptor proteins of rabbit uterine cytosol; in addition, it retained a protein from the cytosols of both rat and rabbit uteri that exhibited an ability to interact specifically with LY117018. The possibility that the LY117018 binding protein, which is distinct from estrogen receptors, may play a role in the antiestrogenic actions of LY117018 is discussed.

Affinity chromatography of estrogen receptors on diethylstilbestrol-agarose.

Diethylstilbestrol was coupled to epoxy-activated agarose yielding an affinity resin which is highly efficient for the isolation of estrogen receptors. This resin, diethylstilbestrol-agarose (DES-agarose), bound two proteins (Mr = 50,000 and 65,000) from rabbit uterine cytosol that show a specific interaction with estradiol. A two step procedure--adsorption on DES-agarose followed by a selective elution with p-sec-amylphenol and NaSCN, yielded highly purified estrogen receptors which can be used in the studies of estradiol-receptor interactions with other cell constituents.

Differential release of estradiol from immobilized estrogen receptors by p-sec-amylphenol: evidence for a functional microheterogeneity of estrogen receptors.

3H-Estradiol-estrogen receptor complexes were adsorbed on a column of heparin-agarose and subjected to a gradient of increasing concentrations of p-sec-amylphenol. At least five peaks of released 3H-estradiol were observed--demonstrating the existence of subsets of heparin-immobilized receptors with different affinities for estradiol. This finding is presented as further evidence for a functional micro-heterogeneity among estrogen receptors. The origin of the observed differences in estrogen receptors and possible relevance of the findings to receptor-mediated responses are discussed.

Release of receptor-bound estradiol by heparin or dextran sulfate in combination with tetracaine.

The rapid release of 3H-estradiol from estrogen-receptor complexes at 0 degrees C is implemented by combinations of the local anesthetic tetracaine and certain sulfated polysaccharides. The simultaneous presence of both classes of compounds was found necessary for the displacement of 3H-estradiol to occur. A limited survey of some sulfated polysaccharides and other anionic compounds for their ability to synergize with tetracaine revealed a specificity for the charge-carrying polysaccharide. The present data support the view that the cooperative interaction of sulfated polysaccharides and certain charged hydrophobic compounds with estrogen receptors determines the ability of the receptors to bind estradiol.

Influence of tetracaine on the structure and function of estrogen receptors.

The local anesthetic agent tetracaine has been shown to relax the affinity of estrogen receptors for estradiol and unidentified ligands in uterine cytosols. This effect is dependent on both the temperature of the exchange reaction and the concentration of tetracaine. Increasing the concentration of tetracaine in the system from 0 to 6 mM while keeping the temperature at 10 C results in a stepwise increase in the fractions of receptors that engage in a rapid exchange. Increasing the temperature of the exchange reaction from 0 to 17 C in the presence of 4 mM tetracaine similarly results in a stepwise increase in the fraction of receptors exhibiting the rapid exchange. The combination of 4 mM tetracaine and 10 C achieves the displacement of [3H]estradiol by unlabeled estradiol from the majority of the receptors in 30 min. In addition, tetracaine appears to relax the specificity of the receptors so as to facilitate the displacement of [3H]estradiol from the receptor by otherwise low affinity ligands, such as tamoxifen, nafoxidine, and 17 alpha-(2',3'-dihydroxypropyl)17 beta-estradiol. Tetracaine also dissociates estrogen receptors from other macromolecules in the cytosol, so that the receptors sediment in low salt sucrose gradients an 4S complexes. The data support the view that estrogen receptors of uterine cytosols are functionally microheterogeneous, possibly by virtue of their interactions with other components of the cytosol, and that set fractions of receptors are transformed to a state with a lower affinity for estradiol by specific concentrations of tetracaine at a given temperature. A concept of receptor action is discussed in which the ability of receptors to mediate the interaction of macromolecules may regulate the activity of multienzyme systems or the function of specific chromatin complexes.