Expression pattern and role of a 60-kilodalton progesterone binding protein in regulating granulosa cell apoptosis: involvement of the mitogen-activated protein kinase cascade.

Progesterone (P4) inhibits both granulosa cells and spontaneously immortalized granulosa cells (SIGCs) from undergoing apoptosis. P4 does so through a plasma membrane-initiated event. It appears that P4's membrane-initiated actions are mediated by a 60-kDa P4 binding protein (P4BP), which is detected by an antibody directed against the ligand binding domain of the nuclear P4 receptor (i.e., C-262). Immunohistochemical analysis revealed that a C-262-detectable protein was first observed in the periphery of a few granulosa cells within early antral-stage follicles. In nonatretic antral follicles, this protein was detected at the periphery of virtually all granulosa cells. In contrast, granulosa cells of atretic follicles lost the distinct peripheral localization of this C-262-detectable protein. This reduction in the membrane localization was also observed by Western blot analysis. To assess the temporal changes in this 60-kDa P4BP during apoptosis, studies were conducted using SIGCs. That this 60-kDa protein is important in mediating P4's action was confirmed by the observation that C-262 but not IgG attenuated P4's antiapoptotic action. Interestingly, the membrane localization of this 60-kDa P4BP was maintained but the ability of P4 to prevent apoptosis was lost within 20 min of initiating the apoptotic cascade. In addition, Erk-1 and -2 phosphorylation (i.e., activity) increased within 20 min of P4 withdrawal. Further, P4 suppressed the increase in the Erk-1 phosphorylation if administered within 5 but not 20 min of initiating the apoptotic cascade. Moreover, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, reduced the percentage of SIGCs undergoing apoptosis in the absence of P4. Because MEK phosphorylates Erk, these observations suggests that 1) the increase in Erk-1 activity is an important part of the apoptotic cascade, 2) P4 promotes granulosa cell viability by modulating the activity of Erk-1, and 3) P4 becomes "uncoupled" from its antiapoptotic signal transduction mechanism within 20 min of initiating apoptosis, even though the membrane localization of the 60-kDa P4BP is maintained.

Lens epithelia contain a high-affinity, membrane steroid hormone-binding protein.

PURPOSE: To describe the serendipitous discovery of a high-affinity, membrane steroid-binding protein (MSBP) in lens epithelial cells and to examine the binding of progesterone to epithelial cell membranes. METHODS: Bovine lens epithelial cells (BLECs) were cultured in media containing 3H-mevalonolactone to examine protein prenylation by mevalonate-derived isoprenes. Cell proteins were divided into insoluble and soluble fractions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and label detected by fluorography. Insoluble proteins were then fractionated on a C18 reversed-phase column. A high-performance liquid chromatography fraction containing a 28kDa 3H-labeled hydrophobic protein was collected, lyophilized, and subjected to SDS-PAGE and the separated proteins transferred to membrane. Protein in the recovered 28-kDa band was submitted for identification by N-terminal sequence analysis. Microsomal membranes prepared from fresh epithelia of intact bovine, rat, and human lens and cultured BLECs were tested for the presence of MSBP by western blot analysis using an antiserum to porcine liver microsomal MSBP. Radiolabeling of MSBP from 3H-mevalonate was confirmed by immunoprecipitation using the same antiserum. 3H-Progesterone was incubated with microsomal membrane from bovine lens epithelia to measure high-affinity binding. Radiolabeled progesterone-protein complexes were trapped on glass filters and radioactivity measured and the binding data subjected to Scatchard analysis. RESULTS: Membrane recovered from BLECs incubated with 3H-mevalonolactone contained a 3H-labeled 28-kDa protein fraction. The N-terminal sequence of the principal protein in this fraction was very similar to that of the recently discovered MSBP. Western blot analysis with antiserum to MSBP indicated the presence of the 28-kDa protein in the microsomal fraction from BLECs and epithelia of bovine, rat, and young human lenses but not in lens fiber cell membrane. Microsomal membrane from intact bovine lens epithelium bound progesterone with high affinity, with disso ciation constant (Kd) at approximately 75 nM and a receptor concentration of approximately 3 picomoles/mg protein. CONCLUSIONS: The lens epithelium contains a 28-kDa membrane protein that can bind progesterone and perhaps other steroid hormones with high affinity. The protein appears to be microsomal and prenylated. The MBSP may mediate rapid nongenomic steroid effects that contribute to steroid-induced cataracts.

Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.

Recognition of a human sperm surface protein involved in the progesterone-initiated acrosome reaction by antisera against an endomembrane progesterone binding protein from porcine liver.

Antisera against a porcine liver endomembrane progesterone (P4)-binding protein inhibited the P4-initiated acrosome reaction (AR) but not the ionomycin-initiated AR of human sperm. Indirect immunofluorescence studies detected antigen in the sperm head that moved during capacitation from a posterior head region to a midhead region. Moreover, the antisera detected a 44.6 kDa protein in western blots of sperm digitonin extracts. These results suggest that a sperm protein with at least partial homology to the liver endomembrane P4-binding protein, is a putative P4-receptor on the sperm plasma membrane involved in the P4-initiated AR.

Localization of a putative progesterone membrane binding protein in porcine hepatocytes.

A putative membrane bound steroid receptor was localized using a peptide specific antibody. Surprisingly, the distribution of immunocytochemical staining in porcine hepatocytes cells provides evidence for the localization to endomembranes (endoplasmic reticulum, Golgi apparatus). Immunofluorescence experiments with HEK cells, which were transfected with a pcDNA3.1 vector containing the coding sequence of the putative progesterone binding protein shows staining within the cells supporting these results. Additionally, 3H-progesterone binding and glucose-6-phosphatase activity as marker enzyme for endoplasmic reticulum were closely correlated in subcellular fractions of porcine liver cells.

Specific interactions of progestins and anti-progestins with progesterone antibodies, plasma binding proteins and the human recombinant receptor.

This structure-activity study compares the affinity of a series of progestins, progesterone metabolites and anti-progestins for a panel of monoclonal antibodies to progesterone, coypu (Myocastor coypus) or guinea pig plasma progesterone-binding proteins (PPBPs) and the human recombinant progesterone receptor A form (PR-A). The compounds tested were progesterone, Promegestone (R5020), Mifepristone (RU486), ZK98,734, Onapristone (ZK98,299), 11 alpha-hydroxyprogesterone, 11 alpha-progesterone hemisuccinate, androsterone, etiocholanolone, 5 alpha- and 5 beta-pregnane-3,20-diones, and 20 alpha- and 20 beta-hydroxyprogesterones. The Ki values for these ligands were determined by competitive binding assays using radiolabelled progesterone as the binding site ligand. For anti-progesterone antibodies (e.g. DB3 and 11/32), only progesterone (3.6-8.8 nM), the 11 alpha-derivatives (1.0-5.5 nM) used to prepare the immunogen and the two 5-pregnanediones (20.9-45.1 nM) were bound with high affinity. For PR-A, high affinity binding was found with receptor agonists (Ki = 1.1-6.2 nM), both 5- and 20-reduced metabolites, and antagonists (0.6-28.0 nM), but not with the 11 alpha-derivatives (950 nM-1.0 microM). In contrast, the PPBPs displayed high affinity interactions with progesterone (3.5-4.2 nM) and both 5 alpha- and 20 alpha-reduced metabolites (2.4-3.4 nM). Binding with the beta-isomers and R5020 was less pronounced (22-170 nM) and there was no evidence of high affinity binding with PR antagonists (> 1.0 microM). Analogs with the 17-keto group did not bind to any of the binders studied. Thus, commonalities among the three types of protein binders were their comparable binding affinities for progesterone (3.5-8.8 nM) and 5-pregnanedione isomers (2.4-330 nM), and a lack of binding for two C17-keto steroids (androsterone and etiocholanolone). The results imply that the tertiary features of the binding domain of these three types of proteins are sufficiently different to result in unique binding structures.

Photoaffinity labeling with progesterone-11 alpha-hemisuccinate- (2-[125I]iodohistamine) identifies four protein bands in mouse brain membranes.

The radiolabeled progesterone (PG) analogue progesterone-11 alpha-hemisuccinate-(2-[125I]iodohistamine) was used to label PG binding proteins in brain membranes from mouse cerebellum. Photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified specific PG binding protein bands 1-4 of 64-29 kDa. Bands 1 and 4 were well resolved on the gel and easily quantified. Preincubation with PG inhibited photolabeling in a dose-dependent manner. The labeling was specific with respect to steroid structure. For band 1, the extent of inhibition of labeling by PG and 3 alpha, 5 alpha-pregnanolone (3 alpha) was pronounced. Other steroids such as testosterone (Tes), estradiol (Est), and corticosterone (Cor) were less effective, whereas pregnenolone sulfate (PS) and cholesterol (Cho) were ineffective. With respect to band 4, Est was the most effective; PG, 3 alpha, and Tes were intermediate; and PS, Cho, and Cor were ineffective. The results describe specific membrane proteins that bind PG (band 1) and Est (band 4).

A specific membrane binding protein for progesterone in rat brain: sex differences and induction by estrogen.

Progesterone conjugated to bovine serum albumin (BSA) was used as a probe to study sex differences and the effects of hormonal status on binding of progesterone to crude synaptosomal membrane preparations (P2) derived from the mediobasal hypothalamic-anterior hypothalamic-preoptic area or the corpus striatum. Binding of 125I-labeled BSA linked to progesterone at the 11 position of the steroid (P-11-BSA) was decreased by competition with unlabeled P-11-BSA or P-3-BSA (in which progesterone is bound to BSA at the 3 position). P-3-BSA displayed higher affinity than P-11-BSA. Hypothalamic and striatal preparations from adult females show high specific binding (60-80%) to the progesterone-BSA conjugate. Specific binding was reduced more than 80% 14 days after ovariectomy. Estrogen treatment (10 micrograms per rat for 4 days) of 14-day ovariectomized rats restored specific binding to levels equivalent to intact females. In contrast, adult males displayed drastically reduced or no specific binding in either tissue. No specific binding was detected after orchidectomy. Estrogen treatment of orchidectomized animals induced specific binding sites similar to those in intact females. Additionally, an affinity probe was developed by linking primary amines on the P-3-BSA conjugate to agarose activated aldehydes in an AminoLink column. A digitoxin-solubilized fraction from female rat P2 cerebellum preparations yielded a single major band after affinity purification with an estimated molecular mass of 40-50 kDa in an SDS/PAGE system after silver stain. These results show a reversible sex difference in the specific binding of progesterone to synaptosomal membrane sites in the central nervous system of male and female rats which is dependent on estrogen.

Properties of proteins binding plasma progesterone in pregnant Cape porcupines (Hystrix africaeaustralis).

The properties of progesterone-binding proteins in plasma of pregnant Cape porcupines were investigated using radiolabelled progesterone and either progesterone or cortisol as competing ligands as well as native plasma and heated (60 degrees C for 30 min) plasma. The results demonstrated that plasma from pregnant porcupines contains corticosteroid-binding globulin, but that it constitutes a significant portion of plasma progesterone-binding proteins only during the early stages of pregnancy. Corticosteroid-binding globulin of porcupines appears to be as heat labile as that of guinea-pigs. Concentrations of progesterone-binding proteins in plasma increased during pregnancy to reach concentrations at the eleventh week that were 25 times higher than those of progesterone; concentrations increased significantly (r2 = 0.88) with the increase in progesterone concentration. The results indicate that plasma progesterone-binding proteins in Cape porcupines (Old World hystricomorph) are similar in composition to those in guinea-pigs (New World hystricomorph).

Adrenocortical nuclear progesterone-binding protein: identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin.

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

The nuclear conversion of pregnenolone to progesterone and subsequent binding to the nuclear progesterone-binding protein in the guinea pig adrenal cortex: a possible regulatory role for the pregnenolone-binding protein.

Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.

Steroid metabolism as a mechanism of escape from progesterone-mediated growth inhibition in Trichophyton mentagrophytes.

It has been shown by us and others that progesterone inhibits the growth of Trichophyton mentagrophytes and that the organism escapes from this inhibition over time. We report here studies which show that escape from growth inhibition is related to the enzymatic transformation of progesterone to polar metabolites. Isolation and identification of the progesterone metabolites confirm the production of 15 alpha-hydroxyprogesterone. In addition, three other metabolites were isolated. Two of these were determined to be 1-dehydroprogesterone and 11 alpha-hydroxyprogesterone. The third metabolite was a 1-dehydro-hydroxyprogesterone, but the location of the hydroxyl group could not be determined unequivocally. Studies using authentic 15 alpha-hydroxyprogesterone, 1-dehydroprogesterone, and 11 alpha-hydroxyprogesterone reveal that these derivatives are significantly less inhibitory to the growth of T. mentagrophytes than progesterone. Pretreatment of organisms with progesterone augments the rate of metabolism and enhances escape. We have described previously a progesterone-binding protein (PBP) in cytoplasmic extracts of T. mentagrophytes and hypothesized that progesterone mediates growth inhibition by binding to the PBP of this organism. The relative binding affinity that progesterone and its metabolites display for PBP correlates with the relative growth inhibitory potency of these compounds. These results suggest that metabolism of progesterone to more polar and less inhibitory compounds, which exhibit lower affinity for PBP, is the mechanism of escape from progesterone-mediated inhibition of growth in this organism.

Sex hormonal changes accompanying loss of massive excess weight.

With adequate weight loss following bariatric surgery, we previously found correction of the clinical gynecologic abnormalities associated with obesity. In this study, sex hormonal changes offered an explanation for the clinical findings.

Properties of progestin-binding protein in benign hypertrophic human prostate.

RU 27987 is a new ligand for progesterone receptor and binds in high affinity to nuclei of target tissues of progesterone. Using this compound, progestin-binding components in the benign hypertrophic human prostate were studied, and compared with those examined with R 5020, a conventional ligand, in the study of progesterone receptor. In cytosols, the binding affinity of RU 27987 was higher than that of R 5020, and the number of maximum binding sites for RU 27987 seemed to be large but correlated well with those of R 5020. The binder for RU 27987 sedimented at 8.6 S, and the binding was specific to progestational steroids, indicating that binding properties of this binder in the cytosols are identical to those for R 5020. Although there was no binding with R 5020 in the nuclear extract, a small amount of specific binding with RU 27987 was detected. However, the cytosol bound with RU 27987 was not retained in DNA Sepharose and no specific binder for RU 27987 in the nuclear extract was observed in a sucrose density gradient centrifugation. From these observations, it was assumed that the nuclear binding observed was attributable to contamination of the cytosolic binder. The results obtained in the present study suggest that the progestin-binding component in the benign prostatic hypertrophy is not the progesterone receptor but a high affinity binder for progestins whose physiological role is not clear at present.

Progesterone-binding protein in the corpus luteum, blood and lymph of sheep.

A protein which binds progesterone but not cortisol was found in luteal cytosol, utero-ovarian venous plasma, ovarian lymph and jugular venous plasma of sheep. The protein was isolated from other steroid-binding activities present in luteal cytosol and plasma by de-adsorption from hydroxyapatite with 40 mM phosphate. In all cases, it bound progesterone at 4 degrees C with an equilibrium affinity constant of the order of 10(6) l/mol, but did not bind cortisol. After chromatography on hydroxyapatite and Sephadex G-200, the protein obtained from utero-ovarian venous plasma had lost much of its steroid-binding activity, but migrated as a monomer of molecular weight 64 000 in polyacrylamide gel. Bovine luteal cytosol is reported to contain two proteins which bind progesterone similarly. In ruminants, these proteins may participate in the biosynthesis and secretion of progesterone from luteal cells and its transport in blood.

Protein binding of progesterone in rat plasma.

The plasma of female rats contained progesterone-binding proteins similar to those found in luteal extracts and plasma of cows and sheep. These proteins were not ovarian in origin. The progesterone-binding activity of ovarian venous and arterial plasma of Day-16-pregnant rats and arterial plasma of rats in dioestrus was resolvable by hydroxylapatite chromatography into three components. The first component was identified as corticosteroid-binding globulin because it bound corticosterone and cortisol as well as progesterone and was not adsorbed to hydroxylapatite. The second and third components were eluted from hydroxylapatite at phosphate ionic strengths of 40 mM and 150 mM and bound progesterone with 99% less affinity. Competition experiments using the second component showed that it bound progesterone specifically. The progesterone-binding components were present in arterial plasma collected from rats which had been ovariectomized 5 days previously and in animals in which circulating concentrations of progesterone had been maintained by subcutaneous injections of progesterone for 6 days after ovariectomy.

The effect of membrane permeability and binding by human serum proteins on sex steroid influx into the uterus.

To determine the effect of such factors as capillary membrane permeability, plasma protein binding, and capillary transit time on the availability of sex steroids to the uterus, the unidirectional influxes of 3H-labeled steroids from the circulation into the uterus were measured in vivo in anesthetized rats using a tissue-sampling, single injection technique. When dihydrotestosterone (DHT), estradiol (E2), and progesterone (P) were injected with Ringer's solution, the tissue extraction was in excess of 80%; hence, membrane permeability did not play a limiting role. With the more polar steroids, corticosterone and cortisol, uterine extraction was less than 40%. Significant inhibition of tissue extraction of DHT and E2, but not P, occurred with the addition of 4% albumin to the injection solution. Human sera containing increasing concentrations of sex hormone-binding globulin demonstrated inhibition of extraction of DHT and E2. Human sera also inhibited P extraction, presumably secondary to the presence of cortisol-binding globulin and orosomucoid. Large concentrations of unlabeled DHT, E2, and P in the injection solutions did not result in competitive inhibition of labeled steroid extraction. Thus, there is no evidence for a carrier mechanism mediating steroid transport into the uterus. When tissue extraction of E2 from Ringer's solution was compared in liver, brain, and uterus, no difference of tissue permeability could be found. Liver consistently had higher tissue E2 extraction than brain or uterus in the presence of human sera. The results are compatible with the influx of albumin-bound E2 into all three tissues and the influx of sex hormone-binding globulin-bound E2 into the liver.

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig.

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.