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J A Katzenellenbogen

Publications and source records attributed to J A Katzenellenbogen.

At least 217 records · Page 12Linked to original sources
PubMed1979

Binding specificity of rat alpha-fetoprotein for a series of estrogen derivatives: studies using equilibrium and nonequilibrium binding techniques.

The binding specificity of rat alpha-fetoprotein (AFP) for a series of estrogen derivatives has been investigated and compared to that of the uterine cytosol estrogen receptor. In addition, several equilibrium and nonequilibrium binding techniques have been examined for their appropriateness in studying AFP-estrogen interactions under different experimental conditions. The ratio of association constants (RAC) of various estrogen derivatives (compared to 17 beta-estradiol; RAC = 100%) were determined using a rapid and convenient miniaturized equilibrium assay that utilizes the partitioning properties of Sephadex G-25. AFP has the highest affinity for estrone derivatives and is especially tolerant of small, nonpolar substituents in the D ring; it binds nonsteroidal estrogens and antiestrogens weakly or not at all. Thus, it has a binding specificity quite unlike that of the estrogen receptor. Several potential affinity labels were studied. Of these, 16-diazoestrone, a photoreactive compound, has the highest affinity for AFP (RAC = 121%). Another equilibrium technique, steady state polyacrylamide electrophoresis, effects a clear separation between AFP and albumin. It is superior to conventional polyacrylamide electrophoresis under conditions where dissociation is rapid. Charcoaldextran adsorption also rapidly removes estradiol that is bound to AFP (t1/2 = approximately 10 min at 0 C). The AFP-estradiol complex appears to be stabilized by adsorption to hydroxylapatite, however, so that hydroxylapatite can be employed in a batchwise manner to assay binding to AFP. This is the most appropriate technique when high concentrations of ligand are used. These studies highlight the marked differences in binding specificity between rat AFP and the uterine cytosol estrogen receptor and indicate the techniques most appropriate for binding measurements under different conditions. This work should facilitate the design of affinity labeling reagents for rat AFP as well.

Animals↗
PubMed1977

Estrogen photoaffinity labels. 1. Chemical and radiochemical synthesis of hexestrol diazoketone and azide derivatives; photochemical studies in solution.

Two photosensitive estrogen derivatives, hexestrol diazoketopropyl ether (5) and hexestrol azide (8a), have been synthesized in radiolabeled form, and their photochemical behavior in solution has been studied. The radiolabeled compounds were prepared in good yields according to improved synthetic procedures; they are stable and were obtained with specific activities in the range of 50-100 Ci per mmol and radiochemical purities in excess of 95%. A simpler model system, phenyl diazoketoprophyl ether, was used to study the photochemical behavior of the diazoketopropyl ether group. Direct irradiation of this compound at 254 nm in methanol led to 33% insertion product (methoxyketone) and 67% Wolff rearrangement product (ester). Irradiation of [3H]hexestrol diazoketopropyl ether (5) in methanol gives mainly nonpolar photoproducts (presumed to be the methoxy ketone and ester); however, irradiation in aqueous medium leads to large amounts of free hexestrol (52%). Photolysis of hexestrol azide (8a) in either methanol or water gives the corresponding amine in low yield as the only identifiable photoproduct.

Affinity Labels↗
PubMed1977

Estrogen photoaffinity labels. 2. Reversible binding and covalent attachment of photosensitive hexestrol derivatives to the uterine estrogen receptor.

The ability of two radiolabeled, photoreactive estrogen analogues, [3H]hexestrol diazoketopropyl ether ([3H]Hex-DKP) and [3H]hexestrol azide ([3H]Hex-N3), to covalently label the uterine estrogen receptor is studied. Lamb uterine estrogen receptor preparations that have been partially purified (ammonium sulfate precipitation, Sephadex G-200 chromatography) and disaggregated by limited trypsinization can be electrophoresed on polyacrylamide gels under conditions where binding activity is preserved. This electrophoretic procedure was used to fractionate the proteins labeled by the two photoreactive estrogen analogues. Prior to photolysis, peaks of radioactivity indicating estrogen specific binding of [3H]-Hex-N3 and [3H]Hex-DKP are evident on the gels, although dissociation of the latter compound is extensive. When preparations of uterine estrogen receptor that contain the photoreactive derivatives are irradiated and then electrophoresed, reversibly labeled proteins can be distinguished from irreversibly labeled ones (covalently bonded), by extraction of the individual gel slices with organic solvents. While no irreversible binding to receptor appears to result from irradiation with [3H]Hex-DKP, irradiation with [3H]Hex-N3 does covalently label the estrogen receptor. The receptor covalently labeled with [3H]Hex-N3 has the same electrophoretic mobility as the unlabeled receptor; the covalent labeling process is estrogen-site specific, and the efficiency of labeling (15-20%) is consistent with the inactivation efficiency of Hex-N3, previously measured by an indirect assay. This is the first example of the labeling of a steroid hormone receptor by photoaffinity labeling.

Affinity Labels↗
PubMed1976

Chemical probes of estrogen action: affinity labeling.

A systemic approach has been taken in the preparation and evaluation of photoaffinity labeling agents for the estrogen receptor from rat and lamb uterus. Several derivatives of estradiol and the nonsteroidal estrogen, hexestrol, containing photoreactive diazocarbonyl or azide functions have been synthesized. The receptor binding affinity of these compounds and their capacity to photointeract with the estrogen binding site (inactivate) can be assayed inderectly by competition assays. Several of the compounds that showed both resonably high binding affinities and inactivation efficiencies have been prepared in high specific activity, tritium-labeled form. Direct binding measurements with these derivatives in unpurified rat uterine receptor preparations, show that while these compounds bind to the receptor, they also show considerable nonspecific binding to nonreceptor proteins. Irradiation of these derivatives in rat uterine cytosol preparations results in incorporation of large amounts of radioactivity into protein in a covalent fashion. The amount of nonspecific labeling is so large, however, that estrogen site specificity (indicated by protection with unlabeled estradiol) cannot be demonstrated. More recently, we have used a partially purified receptor preparation from lamb uterus. The receptor in this preparation has been disaggregated by mild trypsinization and can be electrophoresed in native form. Electrophoretic analysis of the proteins in photolabeled preparations show some covalent incorporation into the receptor region of the gel with one derivative but not with another. The effectiveness of the photoaffinity labeling reagents prepared thus far is assessed, and suggestions are made for the design of new, more effective reagents.

Affinity Labels↗
PubMed1975

Ultraviolet photosensitivity of the estrogen binding protein from rat uterus. Wavelength and ligand dependence. Photocovalent attachment of estrogens to proteins.

Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.

Animals↗
PubMed1975

Iodohexestrols. I. Synthesis and photoreactivity of iodinated hexestrol derivatives.

The five possible ortho-iodinated hexestrols, containing from 1-4 iodines per molecule, have been synthesized as potential iodine-containing estrogenic ligands. After chromatographic separation and purification, each derivative has been fully characterized by spectroscopic methods. [3-H]-3-Iodohexestrol, [131-I]- and [125-I]-3-iodohexestrol, and [125-IA1-3,5-diiodohexestrol have also been prepared. Photolysis of 3,5-diiodohexestrol in methanol results in rapid deiodination to 3-iodohexestrol; further reduction to hexestrol is slower. Photolysis of 3-iodohexestrol in benzene gives 3-phenylhexestrol.

Benzoates↗
PubMed1975

Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo.

The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.

Animals↗