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S S Simons

Publications and source records attributed to S S Simons.

At least 55 records · Page 3Linked to original sources
PubMed1992

Modulation of glucocorticoid induction of stably transfected tyrosine aminotransferase gene constructs involves elements up-stream of the glucocorticoid-responsive element.

Previous studies have documented that the amount of agonist activity expressed by the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) for tyrosine aminotransferase (TAT) induction in two rat hepatoma cell lines (Fu5-5 and HTC) is greater in Fu5-5 cells and could be varied in each cell line with changes in cell density. We have proposed that both phenomena are mediated by the binding of a trans-acting factor, the concentration or activity of which is lower in HTC cells. We have now used DNase-I hypersensitivity studies to identify a possible binding site for this factor at around -3.6 kilobases (kb) of the TAT gene. Fu5-5 and HTC cells were then stably transfected with hybrid constructs either with (3.9TATCAT) or without (2.9TATCAT) this region of the TAT gene fused up-stream of a chloramphenicol acetyltransferase (CAT) reporter gene. High levels of Dex-Mes agonist activity for the induction of CAT activity in Fu5-5 cells were seen only with the 3.9TATCAT construct, indicating that the 0.97-kb region unique to this construct controlled the high levels of Dex-Mes agonist activity. Furthermore, variations in Fu5-5 cell density caused major quantitative changes in the amount of Dex-Mes agonist activity only in cells containing the 3.9TATCAT construct, consistent with the same 0.97-kb sequences also controlling the variations in Dex-Mes agonist activity. Additional studies at high and low cell densities revealed that the modulation of Dex-Mes agonist activity for both the endogenous TAT gene and the transfected TAT/CAT gene was not due to changes in the start site of gene transcription. These studies both support our previous hypothesis that modulation of Dex-Mes agonist activity results from changes in a trans-acting factor and localize a necessary cis-acting element to sequences between -3.9 and -2.9 kb of the TAT gene. These studies, thus, define a potentially new element for glucocorticoid regulation of TAT gene transcription.

Animals↗
PubMed1992

Modulation of transcription factor activity by a distant steroid modulatory element.

Variations in the biological activity of antisteroids, as determined by their percent agonist activity, is a well known but poorly understood phenomenon. For example, in tyrosine aminotransferase (TAT) induction by the antiglucocorticoid dexamethasone 21-mesylate in rat hepatoma tissue culture cells, the percent agonist activity varies with the density of cultured cells. A 21-basepair sequence of the rat TAT gene has now been isolated which confers all of the induction properties of the endogenous TAT gene to homologous and heterologous promoters and genes. We call this 21-basepair sequence, which acts in concert with a trans-acting factor identified by gel shift experiments, a glucocorticoid modulatory element. The changes in induction properties were found to be independent of the fold induction by dexamethasone, thus arguing that the GME does not synergize with the glucocorticoid response element. A model incorporating this new element is advanced which can explain the observed variations of TAT induction and may be generally applicable for the mechanism of action of other steroid hormones.

Base Sequence↗
PubMed1991

Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain.

Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced "super" receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity.

Animals↗
PubMed1991

Association of heat shock protein 90 with the 16 kDa steroid binding core fragment of rat glucocorticoid receptors.

We have recently described a 16 kDa steroid binding core (Thr537-Arg673) of the rat glucocorticoid receptor [Simons et al. (1989) J. Biol. Chem. 264, 14493-14497]. Sedimentation analysis and size exclusion and anion exchange chromatography now suggest that other proteins are associated with the 16 kDa receptor, just as has been seen for the intact 98 kDa receptor. The 16 kDa fragment was also immunoprecipitable with anti-heat shock protein 90 (hsp90) antibody. These results argue that hsp90 binds to the 16 kDa core fragment and directly position the site of hsp90 association between Thr537 and Arg673 of the rat glucocorticoid receptor.

Animals↗
PubMed1991

Dexamethasone 21-(beta-isothiocyanatoethyl) thioether: a new affinity label for glucocorticoid receptors.

The C-21 methanesulfonate ester of the synthetic glucocorticoid dexamethasone (Dex) is an efficient electrophilic affinity label of glucocorticoid receptors and exhibits irreversible antiglucocorticoid activity. In an effort to obtain other affinity labeling steroids with differing biological activities, several new derivatives of Dex were prepared which contained a reactive electrophilic substituent at various distances from the C-21 position. All compounds displayed relatively low affinity for rat glucocorticoid receptors (less than or equal to 8% of that of Dex) in a cell-free competition assay. Nevertheless, one compound, dexamethasone 21-(beta-isothiocyanatoethyl) thioether (Dex-NCS), appeared to be an affinity label by virtue of its ability to block the cell-free exchange binding of [3H]Dex. [3H]Dex-NCS was thus synthesized and reacted with cell-free receptors to give, after analysis on denaturing SDS-polyacrylamide gels, only one specifically labeled species at 98 kDa, which is the molecular weight of authentic rat glucocorticoid receptor. These data directly establish Dex-NCS as a new affinity label for glucocorticoid receptors. Data on the reactivity of Dex-NCS and the stability of [3H]Dex-NCS-labeled receptors suggest that a cysteine SH group has been labeled.

Affinity Labels↗
PubMed1991

Evidence against posttranslational glycosylation of rat glucocorticoid receptors.

The observed M(r) of the rat glucocorticoid receptor on denaturing polyacrylamide gels is 7-11 kDa higher than that deduced from the cloned cDNA sequence of the receptor. Posttranslational modification of the receptor, such as glycosylation, could account for this difference in mol wt. Indeed, several reports have appeared in the literature suggesting that glucocorticoid receptors contain sugar moieties. We have used a variety of methods to determine whether the receptor is glycosylated, i.e., digestion of affinity labeled receptors with various glycosidases, immunoadsorption of receptors after whole cell labeling with [3H]D-glucosamine, and ion exchange and lectin column chromatography of affinity labeled receptors. With each of these methods, there was no evidence for glycosylation of the receptor protein. We therefore conclude that there is no significant amount of either N-linked glycosylation, or O-linked glycosylation with D-glucosamine, of the rat glucocorticoid receptor.

Affinity Labels↗
PubMed1990

Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic.

Both cellular activation signals and exposure to glucocorticoids such as dexamethasone (Dex) cause programmed cell death in T cell hybridomas. When cells were activated in the presence of Dex, however, the degree of killing that was achieved by either stimulus alone was markedly reduced. Dex-induced programmed cell death of normal T cell clones was also prevented by cellular activation. Cyclosporin A (CsA) completely blocked the activation-induced death of T cell hybridomas, but actually enhanced the killing caused by Dex. The addition of CsA to activated T cell hybridomas in the presence of Dex allowed killing to proceed, consistent with ability of CsA to block activation-induced nuclear gene transcription. A number of independent approaches were used to explore the effect of activation on the glucocorticoid signaling/effector pathway. First, RU-486, which binds the glucocorticoid receptor and is a potent competitive antagonist of Dex, did not inhibit activation-induced cell killing. Second, activation of T cell hybridomas did not cause the translocation of the glucocorticoid receptor from the cytoplasm to the nucleus, nor did it prevent the receptor translocation induced by treatment with Dex. Finally, T cell hybridomas were transfected with a plasmid containing the chloramphenicol acetyltransferase (CAT) gene under the control of two tandemly arranged glucocorticoid-responsive elements. Activation of these cells did not induce CAT activity, and did not inhibit the CAT activity induced by Dex. In fact, there was a paradoxical increase in CAT activity when cells were treated with both stimuli. We conclude that cellular activation does not directly utilize the glucocorticoid receptor nor the glucocorticoid pathway when inducing programmed cell death. Furthermore, the ability of activation to inhibit Dex-mediated killing is not due to interference with the classical glucocorticoid signaling pathway, up to and including the initiation of gene transcription. Alternative mechanisms of antagonism, as well as the possible relevance of this phenomenon to the positive selection of self-recognizing thymocytes, are discussed.

Animals↗
PubMed1990

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90.

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.

Animals↗
PubMed1990

Structurally based, selective interaction of arsenite with steroid receptors.

Steroid binding to cognate receptors is of high affinity. However, due to the appreciable homologies in the steroid-binding domains of receptors, this binding is hardly ever totally specific. We have recently obtained evidence that a vicinal dithiol group is involved in steroid binding to glucocorticoid receptors and that these vicinal dithiols are two of the three cysteines in the 16-kDa steroid-binding core. We now report that a comparison of the placement of cysteines in the comparable region of other receptors revealed a lack of similarly closely spaced thiols, which led to the prediction that arsenite would be totally selective in its interaction with glucocorticoid receptors. In fact, 100 microM arsenite inhibited all steroid binding to glucocorticoid receptors while having no effect on the binding of androgen, estrogen, mineralocorticoid, or progesterone receptors. Such total selectivity is not seen for selenite, which is another very potent inhibitor of glucocorticoid binding. This is the first report of absolute selectivity among steroid receptors that is based upon a known structural feature of the receptor protein. This selectivity of arsenite provides the easiest method to date for distinguishing between glucocorticoid and mineralocorticoid receptors and for selectively blocking steroid binding to glucocorticoid receptors in the assays of other receptors.

Amino Acid Sequence↗
PubMed1990

Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function.

Low concentrations of arsenite, but not arsenate, and Cd2+ blocked steroid binding to the glucocorticoid receptors of HTC cells. Inhibition by arsenite was faster and occurred at lower concentrations than for Cd2+. Half-maximal inhibition of [3H]dexamethasone binding was seen after a 30-min preincubation with approximately 7 microM arsenite. The effect of arsenite and of Cd2+ appears to be mediated by a reaction with vicinal dithiols of the receptor as shown by (a) the reversal of arsenite inhibition by much lower concentrations of dithiothreitol (approximately 0.1 mM) than of beta-mercaptoethanol (approximately 10 mM); (b) the ability of both arsenite and Cd2+ to block [3H]dexamethasone 21-mesylate labeling of receptors but not of other thiol-containing proteins; and (c) the known selectivity of arsenite and of Cd2+ for reactions with vicinal dithiols. Arsenite forms a tight complex with these vicinal dithiols since the removal of loosely associated arsenite by gel exclusion chromatography did not reverse the inhibition of steroid binding. The effect of other ions on steroid binding was also examined. Half-maximal inhibition of binding occurred with approximately 5 microM selenite, whereas up to 300 microM Zn2+ was without effect. Much higher concentrations of arsenite were required for effects on unactivated and activated complexes. Arsenite slowly induced a loss of unactivated complexes but rapidly inhibited a portion of the DNA binding of activated complexes. Any effect on activation occurred at arsenite concentrations equal to or higher than those that inhibited DNA binding. In contrast, Cd2+ concentrations similar to those that block steroid binding caused a biphasic loss of unactivated complexes and a marginal loss of activated complexes. This is the first report of effects of arsenite on glucocorticoid receptors. These results confirm directly our earlier hypothesis that steroid binding to rat glucocorticoid receptors involves a vicinal dithiol (Miller, N. R., and Simons, S. S., Jr. (1988) J. Biol. Chem. 263, 15217-15225) and show that arsenite is a potent new reagent for probing receptor structure and function.

Animals↗
PubMed1990

Glucocorticoid receptor binding to calf thymus DNA. 1. Identification and characterization of a macromolecular factor involved in receptor-steroid complex binding to DNA.

Activation of receptor-steroid complexes to a form with high affinity for DNA is a poorly understood process involving multiple components in addition to the holoreceptor. Employing rat HTC cells as the source of glucocorticoid receptor, we show that maximal receptor binding to calf thymus DNA is mediated by a previously unknown small molecular weight factor. This factor can be removed from cytosolic preparations of receptor by gel filtration chromatography. Salt extraction of crude nuclear pellets afforded much larger amounts of a similar DNA-binding activity factor. The cytoplasmic factor and the more abundant nuclear factor were identical on the basis of their similar physical properties. The factor was precipitable in the crude state with (NH4)2SO4 and stable to heat as well as freezing and thawing. Chromatography on DNA-cellulose revealed that the factor itself did not bind to DNA. The factor could be filtered through a Centricon C-3 microconcentrator (molecular weight cutoff approximately 3000) but was excluded from Sephadex G-10 columns. These parameters enable us to determine an apparent molecular weight of 700-3000 for this factor. The presence of large amounts of this factor in nuclei accounts for the previously unexplained observation that, following size exclusion chromatography, more activated complexes bind to nuclei than to DNA. These data indicate that some, but not all, of the activated complexes require factor to be able to bind to DNA. The predominantly nuclear localization of this factor, coupled with its ability to increase DNA binding, attests to the biological relevance of this factor in the whole cell action of receptor-glucocorticoid complexes.

Animals↗
PubMed1990

Glucocorticoid receptor binding to calf thymus DNA. 2. Role of a DNA-binding activity factor in receptor heterogeneity and a multistep mechanism of receptor activation.

In the preceding paper [Cavanaugh, A. H., & Simons, S. S., Jr. (1990) Biochemistry (preceding paper in this issue)], we characterized an apparently identical factor in the cytosol and the nuclear extract of HTC cells that is required for the DNA binding of approximately 43% of the activated receptor-glucocorticoid complexes. In the present study, both those activated complexes that are influenced by this factor and the role of this factor in the process of activation are examined. We find that sodium arsenite inhibits only the DNA binding of those complexes that require factor. Conversely, methyl methane-thiolsulfonate inhibits the DNA binding of only those complexes that are independent of factor. These results provide direct chemical evidence for two populations of activated complexes. Double-reciprocal plots revealed that the increase in DNA binding with endogenous factor occurred by recruiting new complexes for DNA binding as opposed to increasing the binding affinity of existing complexes. These results further suggest that factor associates only with the receptor-steroid complex and does not additionally interact with DNA. A saturable association of factor with complexes was indicated since the amount of available factor in cytosolic solutions decreased after activation of the complexes. Sodium molybdate is known to inhibit the activation of HTC cell receptor-steroid complexes. When factor was added to complexes that had been subjected to activating conditions in the presence of the inhibitor sodium molybdate, no increased DNA binding was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Localization of the vicinal dithiols involved in steroid binding to the rat glucocorticoid receptor.

Our previous studies with the thiol-specific reagent methyl methanethiolsulfonate (MMTS) and the vicinal dithiol-specific reagent sodium arsenite have established that 2 spatially close thiols (i.e. vicinal dithiols) are involved in steroid binding to the intact 98 K rat glucocorticoid receptor. These 2 thiols form an intramolecular disulfide after treatment with low concentrations of MMTS. One of these thiols was proposed to by Cys-656. In an effort to identify both thiols, we have examined the effects of MMTS and arsenite on proteolytic fragments of the receptor, which contain progressively fewer cysteines. MMTS and arsenite are now found to cause the same dithiothreitol-reversible inhibition of steroid binding and affinity labeling of both the 42 K chymotrypsin fragment and the 16 K steroid-binding core fragment of the receptor as was seen for the intact receptor. Characteristic responses include a bimodal inhibition curve for steroid binding after preincubation with MMTS and an inhibition of binding by very low concentrations of arsenite. Low concentrations of MMTS could block steroid binding by forming a disulfide bond between the receptor and a tightly associated, nonreceptor protein. However, no evidence for such cross-linking was observed when intact 98 K receptors, 42 K chymotrypsin fragments, or 16 K trypsin fragments were treated with various concentrations of MMTS, separated on nonreducing sodium dodecyl sulfate-polyacrylamide gels, and visualized by Western blotting with antiheat shock protein 90 or antireceptor antibodies. One of the antireceptor antibodies (aP1) that had been raised against the rat receptor sequence 440-795 was now found to recognize at least 1 epitope in the 16 K core fragment. We conclude that the vicinal dithiols involved in steroid binding are 2 of the 3 cysteines in the sequence of Thr537-Arg673.

Animals↗
PubMed1989

Steroid binding activity is retained in a 16-kDa fragment of the steroid binding domain of rat glucocorticoid receptors.

The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites.

Amino Acid Sequence↗
PubMed1989

Differential modulation of gene induction by glucocorticoids and antiglucocorticoids in rat hepatoma tissue culture cells.

Studies of glucocorticoid and antiglucocorticoid induction of tyrosine aminotransferase (TAT) in two rat hepatoma cell lines (Fu5-5 and HTC) are described. These studies revealed several phenomena that are not consistent with the current models of steroid hormone action: (a) TAT induction occurred at glucocorticoid levels below those required for comparable receptor occupancy in Fu5-5, but not in HTC, cells; (b) the ability of antiglucocorticoids to induce TAT is higher in Fu5-5 than in HTC cells; (c) the values of the amount of TAT agonist activity with the antiglucocorticoid dexamethasone 21-mesylate and of log10 of the dexamethasone concentration required for half-maximal induction of TAT were not constant over time but varied in a linear, reciprocal manner. This modulation was seen for several glucocorticoids and antiglucocorticoids at the level of both TAT enzyme and mRNA but not for two other glucocorticoid inducible genes in the same cells. These results, plus the fact that a similar difference in the concentration required for half-maximal TAT induction in Fu5-5 cells was seen for both glucocorticoids and cyclic AMP, argue that the modulation occurs at some point distal to receptor-steroid complex binding to the biologically active nuclear sites but proximal to translation of TAT mRNA. In order to explain these results, it is pointed out that models involving second messengers are entirely appropriate for steroid hormone action. The participation of a modulated trans-acting factor in such a model may explain the above results.

Animals↗
PubMed1989

Region-specific antiglucocorticoid receptor antibodies selectively recognize the activated form of the ligand-occupied receptor and inhibit the binding of activated complexes to deoxyribonucleic acid.

A synthetic 18-amino acid peptide (Cys500-Lys517) was used to raise polyclonal antibodies in rabbits to the glucocorticoid receptor (GR). The sequence of this peptide is identical to that of residues 500-517 of the rat and 481-498 of the human GR. This sequence overlaps the carboxy-terminal end of the core DNA-binding domain and the amino-terminus of the hinge region of the receptor. Antiserum (AP64) was obtained which recognized both human and rat GR, as determined by immunoblots of receptors immunopurified with authentic anti-GR antibodies, immunoadsorption of both specific [3H]dexamethasone-bound GR and 98K receptors that were specifically covalently labeled by [3H]dexamethasone mesylate, and AP64-induced shifts in the elution position of monomeric [3H]dexamethasone-bound GR from Sephacryl S-300. The specificity of AP64 was demonstrated by the ability of the immunizing peptide, but not a peptide of similar length, to inhibit both the antibody-induced change in elution position from Sephacryl S-300 and the antibody-mediated immobilization of [3H]dexamethasone-bound complexes by protein-A. Further studies indicated that AP64 did not react with native steroid-free GR or with steroidbound (or affinity-labeled) unactivated GR, but did selectively associated with monomeric activated, steroid-bound (or affinity labeled) complexes. AP64 also inhibited the DNA binding of activated complexes in a manner that was specifically blocked by the immunizing peptide. Collectively, these data allow the direct localization of a structural region of the GR that is occluded in the unactivated complex but exposed as a result of activation.

Animals↗
PubMed1988

Steroid binding to hepatoma tissue culture cell glucocorticoid receptors involves at least two sulfhydryl groups.

The presence of a thiol in the steroid binding cavity of glucocorticoid receptors has recently been proved by our affinity labeling of Cys-656 in the steroid binding domain of rat receptors (Simons, S. S., Jr., Pumphrey, J. G., Rudikoff, S., and Eisen, H. J. (1987) J. Biol. Chem. 262, 9676-9680). Studies with the sterically small, thiol-specific reagent methyl methanethiolsulfonate (MMTS) now reveal the involvement of at least two sulfhydryl groups in steroid binding. While the dose-response curves for [3H]dexamethasone binding versus thiol reagent are normally sigmoidal, an unusual bimodal curve is obtained with MMTS in which dexamethasone binding is eliminated at low, but maintained at intermediate, MMTS concentrations. This bimodal dose-response curve demands the involvement of two (or more) thiol groups. Those receptors pretreated with intermediate concentrations of MMTS retain approximately 70% of the initial binding capacity and one-fifth the affinity for dexamethasone. Solutions of this low affinity form of receptor contain essentially no accessible -SH groups, and all of the usual covalent labeling by dexamethasone 21-mesylate of various proteins, including the receptor, is blocked. The facts, that this low affinity form of the receptor is not affected by added iodoacetamide, cannot be produced from the nonsteroid binding form of receptor simply by adding more MMTS, and displays different kinetics of formation than does the nonsteroid binding form of receptor all argue that reaction of the receptor with intermediate and low MMTS, concentrations occurs via different pathways. Nevertheless, the effects of both concentrations of MMTS on the receptor are fully reversible with added dithiothreitol. The kinetics of inhibition of [3H]dexamethasone binding at low MMTS concentrations are independent of receptor concentration, indicating an intramolecular reaction. Collectively these data suggest a model of steroid binding involving two thiols, one of which appears to be Cys-656. Low concentrations of MMTS induce the formation of an intramolecular disulfide, which prevents steroid binding, while the intermediate MMTS concentrations convert both thiols directly to mixed disulfides, and steroid binding persists. Thus, reduced thiols do not appear to be required for steroid binding if the steric bulk of the oxidized thiols is small.

Animals↗