Peter W. Jungblut

Biosynthesis and transformation of microsomal and cytosol estradiol receptors.

Abstract Extracts of uterine microsomes contain a “basic” 3.5 S and an “acidic” 4.5 S estradiol receptor. The smaller “basic” molecule appears to be an early product of receptor biosynthesis. It dimerizes to a “basic” 4.5 S entity on heating. Both the artificial “basic” dimer and the extracted “acidic” 4.5 S molecule are reversibly dissociated by protonation into 3.5 S “basic” and 3.5 S “acidic” subunits respectively. The heat-accelerated formation of stable dimers requires monomer-bound estradiol. The dimers are also dissociated by 2 M urea. High-speed supernatant (cytosol) of uterine homogenates, prepared with either low ionic strength buffer, pH 7.5, or buffered 0.25 M sucrose, contains only “acidic” receptors sedimenting at 4 S and 5 S in sucrose gradients prepared with buffered 0.4 M KC1. The radioactivity sedimenting at 5 S is shifted to the 4 S position by protonation. The reverse shift by proton withdrawal is accelerated by heating. Similar to the estradiol requirement for the formation of stable microsomal 4.5 S dimers, stable cytosol 5 S-estrogen complexes are only derived from estradiolcharged 4 S molecules. In contrast to the microsomal 4.5 S dimer, the cytosol-estradiol 5 S complex is not affected by 2 M urea, which improves the resolution of the two cytosol peaks and avoids the formation of rapidly sedimenting aggregates on heating. The following sequence of receptor forms is indicated as the major in vivo pathway: “basic” microsomal 3.5 S → “acidic” microsomal 3.5 S → cytosol 4 S → cytosol 5 S. Receptor synthesis in the uterus is not necessarily dependent on estradiol since it persists in the uteri of both ovariectomized and ovariectomized/hypophysectomized rats. The administration of estradiol to hormone-deprived animals results in a “depletion-replenishment” sequence of receptor levels, which is caused by irreversible utilization and resynthesis of receptors. Estradiol thus appears to enhance the rate of receptor synthesis.

Activation of transcription-regulating proteins by steroids☆

Abstract The intracellular proteins, which bind steriod hormones with high affinity and specificity have been generally considered as instruments of hormone action. A reversal of assignments might seem a merely semantic exercise, but is indeed in better agreement with experimental evidence identifying ‘receptors’ as transcription-regulating proteins. The series of events in the presence of hormone are: 1. attachment of the steroid to the ‘receptor’ which undergoes a major conformational change when ‘enveloping’ the steroid, 2. dimerization to steroid-receptor: steroid-receptor 3. translocation of the dimer into the nucleus, 4. enhancement of transcription. One product of the latter is ‘receptor’ mRNA, the translation of which initiates within 60–90 min after pulse-administration of steroid. In the absence of hormone, ‘receptor translocation’, degradation and biosynthesis continue to proceed but at a much slower rate. Although these results have been primarily obtained with the estradiol-‘receptor’ system, all other systems seem to follow the same pattern. The molecular mechanism by which enhancement of transcription is achieved is as yet unknown. Its specificity must be quite particular since several steroid-‘receptor‘ systems occur simultaneously within the same cell.

MECHANISMS INVOLVED IN THE REGULATION OF STEROID RECEPTOR LEVELS

Abstract The turnover of steroid receptors comprises: synthesis in the cytoplasm, translocation into the cell nucleus and degradation at a still unknown site. From studies on the oestradiol/receptor system, the following conclusions can be drawn. 1. The in-vivo uptake of oestradiol by target cell nuclei is receptordependent. Steroid and receptor are translocated from the cytoplasm in a 1:1 ratio. A recycling of receptor is undetectable after pulse administration of oestradiol. Receptor replenishment in the cytoplasm is accomplished by synthesis. 2. The nuclear uptake of receptor-in contrast-proceeds also in the absence of oestradiol. Both forms of receptor, monomer and “activated” dimer are present in oestrogen-free nuclei. 3. Oestradiol enhances the “nucleotropy” and the turnover rate of receptor. 4. Oestrogenicity and antioestrogenicity are apparently linked to effects exerted on the “nucleotropy” of receptor and its ability to interact with the relevant nuclear structures.

Electron spectroscopic imaging of antigens by reaction with boronated antibodies.

Two small homogeneous markers for electron spectroscopic imaging (ESI) containing eight dodecaborane cages linked to a poly‐α,ε‐l‐lysine dendrimer were synthesized; one of these was made water soluble by the attachment of a polyether. The markers were coupled to the sulfhydryl group of (monovalent) antibody fragments (Fab′) by a homobifunctional cross‐linker. While the coupling ratios of the poorly water‐soluble compound did not exceed 20%, the polyether‐containing variant reacted quantitatively. Its suitability for immunolabelling was tested in a study of the mechanism of the transcellular transport of an administered heterologous protein (bovine serum albumin, BSA) through ileal enterocytes of newborn piglets by endocytotic vesicles in comparison to conventional immunogold reagents. The post‐embedding technique was employed. The boronated Fab′ gave rise to considerably higher tagging frequencies than seen with immunogold, as could be expected from its form‐ and size‐related physical advantages and the dense packing of BSA in the vesicles. The new probe, carrying the antigen‐combining cleft at one end and the boron clusters at the opposite end of the oval‐shaped conjugate, add to the potential of ESI‐based immunocytochemistry.

The C-terminal half of the porcine estradiol receptor contains no post-translational modification: determination of the primary structure.

The C-terminal part of ligand filled porcine estradiol receptor extending from H267 to I595 was isolated by adsorption to the monoclonal antibody 13H2, subjected to cleavage by CNBr, o-iodosobenzoic acid and endopeptidase Lys-C as well as other proteases, both in the native and the denatured state. The overlapping peptides produced were separated by reverse phase HPLC and sequenced by Edman degradation, lacking T570-M581 in domain F. We found no evidence of post-translational modification; the native fragment is not glycosylated and the tyrosyl residues in domain E (aa 328, 331, 459, 526, 537) and F (aa 582, 583) are not phosphorylated. In addition, all serine and threonine PTH derivatives were obtained in normal yields. The amino acid sequence of the fragment corresponds in full with that derived from the cDNA. The complete cDNA-derived sequence codes for a polypeptide of 595 amino acids with a calculated mass of 66,357 Da. The high degree of homology between species in domains C and E is shared by the porcine receptor.

Alterations in the subcellular distribution of 17β-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17β-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2–4 μm diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.

Immunoreactivity of the core of estrogen receptors found in different subcellular compartments of target cells from a variety of mammalian species

A goat antiserum was raised by immunization with the endoglycosidase fission product of porcine estrogen receptor. Its IgG fraction was employed for ascertaining the interrelationship of receptor forms extracted from three subcellular compartments of target cells and for comparing receptors from various species. Unspecific alignments of (non-immune) IgGs giving rise to soluble complexes were avoided by removal of the receptor entity responsible with carrier-attached trypsin. Coincubation of the estradiol-tagged tryptic receptor cores and immune IgG resulted in the formation of labelled precipitates and in the release of estradiol from its binding site, in every instance tested. A minimum of three common antigenic determinants must therefore exist not only on all porcine receptor forms, but also on human, ovine, bovine, rabbit, guinea-pig and rat estrogen receptors, pointing to a high degree of phylogenetic conservation.

IDENTIFICATION OF TARGET CELLS BY IMMUNOHISTOCHEMICAL DETECTION OF COVALENTLY REARRANGED ESTRADIOL IN REHYDRATED PARAFFIN SECTIONS

Abstract Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.

Studies on the involvement of lysosomes in estrogen action, I. Isolation and enzymatic properties of pig endometrial lysosomes.

Pig endometrium cells, collected by curettage and homogenized in an all-glass Potter Elvehjem homogenizer, gave a considerably higher yield of intact mitochondria and lysosomes than homogenates of whole uterus obtained with the Ultraturrax or the Parr bomb. After homogenization of the cells and subfractionation in the presence of Mg2, mitochondria and lysosomes equilibrated at the same modal density in isopycnic centrifugation. Homogenization and subfractionation in buffers devoid of divalent cations and containing EDTA resulted in a decrease in the buoyant density of mitochondria, allowing for a separation from lysosomes. The pH optima and the specific activities of two mitochondrial enzymes and eight hydrolyases used as marker enzymes were determined. The morphological characteristics of fractions were established by electron microscopy. Preliminary results indicate an involvement of lysosomes in steroid metabolism rather than in steroid and receptor translocation into the nucleus.

Binding of Steroids by Tissue Proteins Steroid hormone “receptors”

Publisher Summary This chapter discusses the binding of steroids by tissue proteins steroid hormone “receptors”. Estrogens are among the biologically active substances that act at the cellular level in low concentrations. They have been compared earlier with cardiac glycosides for which pharmacologists proposed a sort of hit and run mechanism of action, because the minute amounts present could not be detected with the techniques available. With the advent of radioactive compounds of high specific activity, this concept had to be revised. The experiments of Jensen and Jacobson extended and confirmed by numerous laboratories, leave no doubt that estradiol is selectively accumulated and retained by the target cells.