Bound steroids

The first crystal structure of the LED of the AR in complex with metri-bolone (R1881) was solved by Matias and colleagues in 2000 [38]. The LED has a similar three-dimensional structure to the other agonist-bound steroid receptors, namely the ERa, ERj6 and the PR [38]. The fact that all steroid hormone receptors bind similar hormone response elements (HREs) stands in sharp contrast to the specific activities elicited by application of the steroid... 

An example of side-chain fimctionalizahon is the attachment of tetraphenyl-porphyrin carboxylic acid to a fullerene bound steroid (214) via standard EDCl coupling with the hydroxy group of the steroid (Scheme 4.36) [230]. 

Another complication is the possible interaction of inorganic sulfur species with A - and A -sterols. The occurrence of thiosteroids and of sulfur-bound steroid units in high-molecular-weight fractions of sediments and oils has been observed recently (15.16.20,26). The thiosteroids with the thiophene ring condensed to the D-ring almost exclusively possess the 140(H) configuration (26). Thiosteroids with a thiophene unit in the side chain mainly... 

Secondly, in the case of the bound steroids it was possible to locate the position of the (poly)sulfide linkages in these structural units Le. mainly at position 3 of the steroid units. This strongly supports the argument that functionalised lipids are incorporated into these macromolecules by reaction of H2S and/or polysulfides with functionalities, since sterols or corresponding sterenes are the only likely precursors for the steroid units in these macromolecules. 

Substituting [B] for the concentration of bound steroid, [F] for the concentration of free steroid, and [Rtot] = [R] + [SR] for the total number of receptor sites, this relationship can be rewritten as ... 

In one procedure [24] cells are rapidly centrifuged through an oil layer with a density in between the density of cells and medium. In this way cells and medium are not only separated within seconds, preventing redistribution of bound steroid, but also steroid adhering to the cell surface is removed by the lipophilic separation layer. 

Blossey EC, Cannon RG, Ford WT, Periyasam YM, Mohanraj S, Synthesis, reactions and carbon-13 FT NMR spectroscopy of polymer-bound steroids, J. Org. Chem., 55 4664 1668, 1990. 

The chemistry of the coupling reaction of the allenic secosteroids to isomerase has been under investigation by Penning and Talalay (82, 83, 84) who report that the site of attachment is in the tetrapeptide sequence 55-58. Identification of the actual site has been hampered by a lability of the bound steroid to both low and high pH. 

A further example of the utility of this approach involved the polymerization and manipulation of cholanic-acid-derived xanthate 59 (Scheme 14) [10]. Treatment of xanthate 59 with 15 equivalents of styrene provided polymer-bound steroid 60 in 85% yield. In this case a spacer unit was incorporated between the polymer and the substrate. The substrate was then modified at both the xanthate and carboxylic acid termini to provide N-cyclopropylamide 61. Cleavage from the polymer support was achieved using KOH in methanol and THF, and provided amide 62 in 62% yield. 

Scheme 14 Synthesis of a polymer-bound steroid derivative by Zard [10]...

DNA. Steroid receptors have been reported in mitochondria (17) and cell membranes, although it is not yet clear whether all of these receptors are the same as the intracellular steroid receptors (16, 18-22 vs. 23). Some of the membrane-bound receptors for steroids are G protein-coupled receptors (24—27). A recent report suggests that membrane-bound steroid receptors can interact with, and augment the transcriptional activity of, the intracellular receptors (24). Finally, steroids can bind to nonreceptor molecules such as enzymes and transport proteins (see above), which may have yet undiscovered consequences. 

Any radioimmunoassay analysis relies upon the efficiency of the separation of the unbound (free) antigen or hapten from that bound to antibody. When using immunoassay to estimate low molecular weight compounds such as steroids, the problem is perhaps somewhat simpler than in protein immunoassay. Because of the large difference in molecular weight between steroid haptens and their appropriate antibodies, relatively simple methods of separation, such as dialysis, can be used. Although nonspecific precipitation of the antibody-bound steroid by ammonium sulfate and polyethylene glycoH are widely used, the utilization of second-anti-body immunoprecipitation of the steroid-antibody complex is not com-... 

Antibody-bound steroid was adsorbed to hydroxyapatite after addition to the incubation medium as a suspension or a solid powder. Free and antibody-bound steroid can also be separated in small columns of hy-droxyapaptite, eluting antibody-bound steroid with a continuous, increasing concentration of phosphate buffer, pH 7.0 (see Fig. 1). 

Evaluation of Hydroxyapatite as an Adsorbent of Antibody-Bound Steroid. ... 

Investigations have shown that the use of hydroxyapatite appears to be a very satisfactory procedure for the separation of free and antibody-bound steroids and other low molecular weight compounds (see the table). The adsorption of antibody-bound steroid appears to be independent of time in contact with hydroxyapatite (up to 1 hr), temperature (4-37°), and amount of hydroxyapatite added. Buffers of pH > 7.5 appear to prevent adsorption to varying degrees, and phosphate buffers elute antibody-bound steroid from hydroxyapatite (see Fig. 3). However, Tris-HCl buffers up to molarities of 0.1 M have no appreciable effect on the adsorption of antibody-bound steroid. No significant effects on the crossreactivity of the various antisera used has been noted. 

Fig. 3. Effect of increasing concentrations of phosphate buffer, pH 7.0, on the adsorption of antibody-bound steroid. pH]Testosterone was incubated with antibody without added unlabeled testosterone. Hydroxyapatite suspensions in phosphate buffers of different molarities were added tubes were shaken and centrifuged, and the supernatants were counted. Phosphate buffer strengths recorded are the final concentrations after addition of the hydroxyapatite suspension.

Plasma has been treated in various ways in attempts to identify and remove the interfering component. Dialysis, heat treatment (80° for 20 min), and oxidation with periodate have had no effect. Precipitation of plasma proteins with trichloroacetic acid or perchlorate, and subsequent dialysis, produced a dialyzate that had no appreciable effect on hydroxyapatite. This approach is impractical, however, because it cannot easily be applied to plasma immediately prior to assay. So far, we have not managed to find a simple solution to this problem that will enable hydroxyapatite to be used to separate free and antibody-bound steroid in the presence of significant concentrations of nonextracted plasma. 

Hydroxapatite has proved to be very useful for the separation of free and antibody-bound steroid in the immunoassay of various steroids. Successful immunoassays have been set up, using hydroxyapatite, for the estimation of plasma testosterone, estradiol-17/3, 17a-hydroxyproges-terone, and progesterone. No steroid so far examined has been found to be bound to hydroxyapatite. Some other low molecular weight compounds, for which immunoassays are available, have also been found not to be bound to hydroxyapatite (see the table), and it may therefore be possible to use hydroxyapatite in immunoassays for compounds other than steroids. 

Kohnen M. E. L., Sinninghe Damste J. S., Baas M., Kock-van Dalen A. C., and de Leeuw J. W. (1993) Sulphur-bound steroid and phytane carbon skeletons in geomacromolecules implications for the mechanism of incorporation of sulphur into organic matter. Geochim. Cosmochim. Acta 57, 2515-2528. 

By choosing suitable reaction conditions, it is possible to use the system as a very sensitive assay technique. In the presence of a fixed amount of binding protein, the amount of steroid will determine the ratio of unbound steroid to steroid-protein complex at equilibrium. By adding a tracer amount of labelled steroid to the system, a simple means of determining this ratio is available, and hence, by reference to a standard curve, the amount of steroid can be found. In practice, this usually requires separation of the unbounded [St] and bound [St Prot] fractions, and then isotope counting of one or other fraction. Separation of unbound and bound steroid may be... 

In the equilibrium dialysis/ultrafiltration method for determining free testosterone in blood, a sample is first equilibrated with radioactive testosterone. Free steroid is then separated from bound steroid by filtration through an anisotropic, hydrophilic ultrafiitration membrane. The driving force for ultrafiltration is provided by centrifugation at 1000 to 2000 xg. Filtrate containing free steroid collects in the filtrate cup, whereas protein-bound steroid remains above the filter. Radioactivity in the filtrate is a measure of... 

Wu CH, Mennuti MT, Mikhail G. Free and protein-bound steroids in amniotic fluid of midpregnancy. Am J Obstet Gynecol 1979 133 666-72. 

Application of gel phase NMR to organic solid phase synthesis was first reported by Manatt in 1980 (12). Jones and Leznoff extended this work to show that13 C NMR data could be obtained from polystyrene-supported pheromones and a wide variety of organic substrates (13). The application of 13C gel phase NMR to the study of functional group interconversion of polymer-bound steroids has also been reported (14). High-resolution H gel phase NMR has been reported for an octapeptide (15). The NMR data were obtained by use of a deconvolution method to enhance the spectral resolution. 

BE C Lossey, RG Cannon, WT Ford, M Periyasamy, S Mohanraj. Synthesis, reactions, and 13C FT NMR spectroscopy of polymer-bound steroids. J Org Chem 55 4664-4668, 1990. 

If the receptor-bound steroid is also in its minimum energy conformation, then it should be possible to compare the crystallographically observed structures of a series of steroids that compete for a specific binding site and determine the structural... 

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