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H J Johnson

Publications and source records attributed to H J Johnson.

At least 37 records · Page 2Linked to original sources
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↗