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The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.

Although the pseudohalide thiocyanate (SCN−) is the preferred substrate for eosinophil peroxidase (EPO) in fluids of physiologic halide composition, the product(s) of this reaction have not been directly identified, and mechanisms underlying their cytotoxic potential are poorly characterized. We used nuclear magnetic resonance spectroscopy (NMR), electrospray ionization mass spectrometry, and quantitative chemical analysis to identify the principal reaction products of both the EPO/SCN−/H2O2 system and activated eosinophils as roughly equimolar amounts of OSCN−(hypothiocyanite) and OCN− (cyanate). Red blood cells exposed to increasing concentrations of OSCN−/OCN− are first depleted of glutathione, after which glutathione S-transferase and glyceraldehyde-3-phosphate dehydrogenase then ATPases undergo sulfhydryl (SH) reductant-reversible inactivation before lysing. OSCN−/OCN− inactivates red blood cell membrane ATPases 10–1000 times more potently than do HOCl, HOBr, and H2O2. Exposure of glutathione S-transferase to [14C]OSCN−/OCN− causes SH reductant-reversible disulfide bonding and covalent isotope labeling. We propose that EPO/SCN−/H2O2reaction products comprise a potential SH-targeted cytotoxic system that functions in striking contrast to HOCl, the highly but relatively indiscriminantly reactive product of the neutrophil myeloperoxidase system.

Follicle-stimulating hormone (FSH) is a glycoprotein hormone composed of two different subunits designated FSH-alpha and FSH-beta. Using synthetic peptides corresponding to the primary structure of human (h) FSH-beta subunit, we previously identified two regions of the beta-subunit, hFSH-(33-53) and hFSH-(81-95), as receptor binding regions. In this report, we tested the ability of synthetic peptides to interact with hFSH-alpha-subunit. Synthetic peptides corresponding to hFSH-beta-(11-25), (41-55), (51-65), and (101-111) were able to bind specifically radioiodinated hFSH-alpha-subunit, suggesting that they represent regions of interaction with the alpha-subunit. These experimental results were in agreement with the location of alpha-subunit contact regions predicted by sequence analysis. Peptides of hFSH-beta-subunit showing maximum specific binding to the alpha-subunit were those possessing minimum interaction with receptor whereas those not binding to alpha-subunit corresponded to regions shown to interact with receptor (hFSH-beta-(33-53) and hFSH-beta-(81-95]. The hFSH-beta-subunit, therefore, seems to have two discontinuous receptor binding regions flanked by three alpha-subunit contact regions.

Follitropin (FSH) receptors were solubilized from pure light membranes of bovine calf testis, using an optimum detergent to protein ratio of 0.01. The soluble FSH receptor fraction was gel filtered through Sepharose 6B to isolate an active fraction (6B-Fr-1) which behaved as a complex of FSH receptor and Gs protein. The 6B-Fr-1 was concentrated by ultrafiltration and further purified by sequential Sepharose 4B gel filtration, DEAE-cellulose chromatography (to separate the receptor from Gs protein), and wheat germ lectin affinity chromatography. The purified receptor had an FSH-binding capacity of approximately 3.47 nmol/mg of protein with a Kd of 1.9 X 10(-10) M. Yield was 526 micrograms/11.5 kg tested. Radioiodinated, as well as unlabeled purified FSH receptor, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single major band of Mr approximately 240,000. This band was not affected by 8 M urea treatment prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but treatment with dithiothreitol induced the loss of the 240-kDa band, with appearance of an Mr approximately 60,000 band. The availability of highly purified, stable FSH receptor should allow direct studies on its structure-function relationships.

Pituitary and placental glycoprotein hormones are heterodimers with alpha-subunits of identical primary structure, but dissimilar beta-subunits. Regions of structural similarity between the beta-subunits might be involved in interaction with the homologous alpha-subunits, and regions of structural dissimilarity could, therefore, be candidates for receptor interactions. A restrained matrix dot-plot analysis identified hFSH-beta-(8-32) and hFSH-beta-(55-65) as candidates for interaction with alpha-subunit. Therefore, by subtraction, hFSH-beta-(33-54) and hFSH-beta-(66-111) seemed candidates for regions of interaction with receptor. In a previous report we demonstrated that hFSH-beta-(33-53) represented a receptor-binding region of hFSH-beta. Analysis of structural parameters (flexibility, surface probability, secondary structure prediction, etc.) indicates similarities between hFSH-beta-(33-53) and hFSH-beta-(85-95), suggesting the latter might be the component of hFSH-beta-(61-111) interacting with the receptor. Testing of 11 synthetic peptides, corresponding to the primary structure of hFSH-beta, demonstrated that hFSH-beta-(31-45)-peptide amide, were unique in ability to inhibit 125I-follicle-stimulating hormone binding to receptor. hFSH-beta-(81-95)-peptide amide also stimulated estradiol biosynthesis in Sertoli cell cultures. The correlation between binding inhibition and surface probability, flexibility, and predicted secondary structure (alpha, extended, and turn) was highly significant (R2 = 0.87, p less than 0.0001). Regression significance for these parameters, taken individually, were very poor. Receptor-binding regions, therefore, appear to be characterized by a particular and complex arrangement of secondary structure motifs, surface probability, and flexibility.

Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate that the calf testis FSH receptor has a multimeric structure containing at least one 48-kDa subunit and suggest the presence of other nonidentical receptor subunit proteins.

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.

Attempts have been made to solubilize gonadotropin receptors from rat testes by extraction with ethanol. This approach is in contrast to earlier studies which have generally employed nonionic detergents such as Triton X-100 or Lubrol PX for receptor solubilization. Extraction of homogenates of mature rat testes with 30% (v/v) ethanol in 0.01 m phosphate pH 7.5 (37°) causes solubilization of factors which appear to interact specifically with iodinated human luteinizing hormone (hLH) or follicle-stimulating hormone (hFSH). Interaction of such ethanol-soluble factor (or factors) with hFSH and hLH was seen with the initial ethanol extract, after heating of the ethanol extract (5 min at 100°) to remove easily denatured contaminants and after fractionation of the heated ethanol extract by gel filtration through Sephadex G-100. Evidence for such interaction was obtained from three different types of experiments. In the first it was shown that the solubility of free, iodinated hFSH or hLH in polyethylene glycol (Carbowax 400, 47% v/v) was enhanced by ethanol-soluble factor (or factors). This phenomenon was interpreted as a reflection of direct interaction of components of the ethanol-soluble testicular extract with hLH or hFSH, and is in contrast with results obtained using detergent extracts of testicular and ovarian tissue wherein polyethylene glycol is used to precipitate the putative hormone-receptor complex. Second, when applied to a column of Sepharose or Sephadex G-25 which have been previously equilibrated with buffer containing radioactive hFSH or hLH until a constant amount of radioactivity emerged from the column, the ethanol-soluble factor concentrated radioactivity upon elution. Passage of ethanol-soluble factor through a similar column equilibrated with radioiodinated human serum albumin produced no such concentration of radioactivity, nor did filtration of extracts of rat testis not containing ethanol. Finally, the interaction of hFSH and hLH with unheated, heated, and Sephadex G-100-fractionated ethanol extracts of testicular tissue were examined by circular dichroism spectroscopy. The circular dichroism spectra of mixtures of hLH or hFSH with each of these three types of ethanol-soluble extracts differed significantly in detail and in sum from the circular dichroism spectra obtained when components of the mixture were analyzed separately. The most pronounced differences in circular dichroism spectra occurred in the near ultraviolet region (250 to 350 nm). In the far ultraviolet region (200 to 250 nm), the sums of the circular dichroism spectra of hLH or hFSH and the various ethanol-soluble extracts tested, were additive. Differences in circular dichroism spectra of mixtures of hormone- and ethanol-soluble factor (or factors), compared to circular dichroism spectra predicted on the basis of analysis of individual components of the mixture, are considered indicative of hormone-factor interaction. Of the seven fractions obtained following gel filtration of heated ethanol-soluble factor through Sephadex G-100, only three gave evidence of interaction with FSH or LH. Of these, the fractions which interacted most strongly with FSH or LH had average distribution coefficients of 0.19 and 0.29, respectively. Based upon the relationship between the average distribution coefficient of standard proteins and the log of their molecular weights, these correspond to approximate molecular weights of 67,000 and 22,500, respectively. The possibility cannot be excluded, however, that the circular dichroism active species is either not protein in nature, or represents an active component formed in a complex with a protein, such as a lipid. Although the ethanol-soluble factor appears necessary for binding of FSH or LH by tissue receptors, the gonadotropin-receptor complex, once formed, can no longer be dissociated with ethanol under conditions found effective for solubilization of the factor (or factors). This may indicate that the ethanol-soluble factor (or factors) is a necessary intermediate for hormone-receptor interaction, but is not required for maintenance of the complex once it is formed.

Homobifunctional cross-linkers were utilized to characterize high affinity (Ka = 2.2 X 10(-10) M-1) follitropin (FSH) receptors in immature bovine testis. Following the formation of radioiodinated human FSH (125I-hFSH)-receptor complexes, the membranes were solubilized with Triton X-100 or beta-octyl glucoside and the supernatants from ultracentrifugation (220,000 X g) subjected to gel filtration (Sephadex G-200) to separate the labeled hormone-receptor complexes from the unbound 125I-hFSH. The appearance of a high molecular weight (greater than or equal to 200,000) radioactive component in the elution profile was abolished when an excess of unlabeled hFSH was included in the initial incubation. After concentration by ultrafiltration, the 125I-hFSH-receptor complex, as well as the free hormone, was treated with a variety of chemical cross-linkers and subjected to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Bands of Mr = 65,000 and 83,000 observed in the autoradiograph of the hormone-receptor complex was not present in autoradiographs of free 125I-hFSH, nor were they present when an excess of unlabeled hFSH was included in the initial binding incubation mixtures. The 65,000 and 83,000 Mr bands were, therefore, considered to represent cross-linked complexes of labeled hFSH (Mr = 38,000) or its subunits (hFSH alpha, Mr = 16,000; hFSH beta, Mr = 21,000) and components of the FSH receptor. The bands were observed on autoradiographs when the extraction of the membranes was performed with either Triton X-100 or beta-octyl glucoside and when cross-linking was accomplished with disuccinimidyl suberate, ethylene glycol bis(succinimidyl succinate), or bis[2-(succinimido oxycarbonyl)oxyethyl]sulfone. The Mr of the native FSH receptor in the calf testis has been estimated at 146,000. Our studies demonstrate the multimeric nature of the FSH receptor. However, FSH is also composed of subunits, so that due to the complexity of the system, it was not possible to arrive at a precise assessment of the Mr or quaternary structure of the receptor subunits.

A rat testes tubule tissue receptor assay was utilized to study the kinetics of combination of the hormone-specific β subunit of human follicle-stimulating hormone (hFSH) with the α subunits of hFSH, human luteinizing hormone (hLH), bovine luteinizing hormone (bLH), and human chorionic gonadotropin (hCG). In this competitive protein-binding assay, 125I-hFSH is the radioligand and FSH activity is measured by the ability of the test substance to inhibit uptake of the radioligand by the rat testes tubule receptors. Rate data for combination of subunits were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. The time course for the appearance of FSH activity upon incubation of the various subunit combinations was neither clearly first or second order, although each simple mechanism could be fitted to the experimental data. We conclude that at least one kinetically distinguishable state follows formation of the initial α/β subunit complex and that this complex has a binding affinity for its receptors in rat testes tubules which is much less than those of subsequent states. Hybrid molecules of bLH-α/hFSH-β, hLH-α/hFSH-β, and hCG-α/hFSH-β were formed at differing rates, but each was more rapid than the combination always gous hFSH-α/hFSH-β pair. Maximum combination always occurred by 1440 min of incubation. Hybrid molecules formed after this time were tested for FSH activity by the in vitro rat testes tubule receptor assay and results compared with FSH activity as measured by a classical in vivo bioassay. The in vitro activity of hFSH-α/hFSH-β, hLH-α/hFSH-β and hCG-α/hFSH-β hybrid molecules was from 2 to 4 times greater than their activity as measured in vivo. The in vitro activity of these hybrids was similar to that of intact hFSH, but the activity of the hybrid molecules in vivo was less than that of intact hFSH. Of particular interest were the properties of the hybrid molecule formed between bLH-α/hFSH-β. This combinant had an in vitro activity which was 2.8 times greater than that of purified hFSH. The activity of the bLH-α/hFSH-β hybrid as measured by the in vivo bioassay, however, was only 0.14 times the activity of purified hFSH. The ratio of activity in vitro to in vivo for this artificial FSH molecule was 19.8. It is suggested that the bLH-α/hFSH-β hybrid has a greater affinity than native hFSH for receptors in rat testes tubules, but that expression of in vivo biological activity is depressed probably because of factors related to its more rapid clearance from the circulation.

The quaternary structural relationships between subunits of the follitropin (FSH) receptor were determined through the use of the reversible, homobifunctional, chemical cross-linking reagent bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) after formation of covalent 125I-azidobenzoyl-FSH-receptor subunit complexes by photoaffinity labeling. This experimental approach resulted in the formation of high molecular mass complexes (116, 172, and 320 kDa) as detected by autoradiography. After reversal of the BSOCOES cross-links, a second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these complexes demonstrated that two lower molecular mass complexes, 64 and 84 kDa, identified previously as specific components of the FSH receptor by photoaffinity labeling alone (Smith, R. A., Branca, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 14297-14303) were contained within the 116- and 172-kDa complexes. In addition, the 116-kDa complex was not found to be a component of the 172-kDa complex. Since the high molecular weight complexes have molecular weights large enough to contain additional unlabeled proteins, these data indicate the possibility that there are several distinct FSH receptor subunits. Furthermore, the observation of the 320-kDa band, taken together with the observed structural relationships, suggests that the FSH receptor may contain two each of three distinct subunits with approximate molecular weights of 32,000, 48,000, and 86,000, respectively.

Subunits of bovine pituitary luteinizing hormone (LH) have been isolated by countercurrent distribution, using a modification of the system employed by Papkoff and Samy (Biochim. Biophys. Acta, 147, 175 (1967)) in studies on ovine LH. The subunit having a low partition coefficient was designated C-1, and that with a high partition coefficient, C-2. Gel filtration data indicated intact LH to have a Stokes radius of 2.76 mµ and a molecular weight of 29,258. Each subunit had a Stokes radius of 2.28 mµ and a molecular weight of 14,609. Diffusion coefficients and frictional ratios were also calculated. There were significant differences in chemical composition between the subunits. Subunit C-2 had 2.64 times as much proline and 3.19 times as much leucine as did C-1. The lysine to arginine ratio for C-1 was 3:1, while for C-2 it was 1:3. In addition, the ratio of hydrophobic to hydrophilic amino acids for subunit C-2 was practically double that found in C-1. No NH2-terminal amino acids could be found for either subunit, but the COOH-terminal amino acids appeared to be serine for C-1 and leucine for C-2, on the basis of carboxypeptidase A digestion and hydrazinolysis studies. Each subunit possessed definite biological and immunological activity. The biological activities of C-1 and C-2 were 30% and 3.9% of intact LH. The immunological activities of C-1 and C-2 were 24% and 2% of intact LH. It was possible to achieve a reassociation of the subunits by incubation in 0.01 m phosphate buffer, pH 7.0, for 16 hours at 40°. This reassociation could be demonstrated by gel filtration and biological measurements. The physical properties of the recombined C-1 plus C-2 subunit molecule were essentially identical with those of intact LH. When combined in a 1:1 ratio by weight, the biological potency of the recombined molecule was augmented 3.35 times over that predicted on the basis of individual subunit potencies. Immunological potencies of the recombined molecule were augmented 3.42 times over predicted values. When compared to intact LH, recovery of biological activity was 57% and immunological activity, 44%.

Some properties of the interaction between radioiodinated human follicle stimulating hormone (125I-hFSH) and its specific receptors in preparations of rat seminiferous tubule homogenate are described. Specific binding of 125I-hFSH by seminiferous tubule homogenate was appreciably greater than to Leydig cell homogenate of rat testes. The uptake of 125I-hFSH was hormone specific and uptake inhibition seen with purified human luteinizing hormone (hLH) could be attributed to residual FSH activity in the latter. While biologically active FSH inhibited the specific uptake of 125I-hFSH by seminiferous tubule homogenate, biologically inactive FSH, denatured by heat, did not. The system could also be used to follow formation of intact FSH from its α and β subunits, thus further validating the relationship between specific binding and biologic activity. The uptake of 125I-hFSH was tissue specific, since negligible specific binding was seen when a variety of rat tissues other than seminiferous tubule homogenate were examined. The uptake of 125I-hFSH was 1.6-fold greater in seminiferous tubule homogenate derived from testes of mature rats compared to immature (16-day-old) rats. Specific 125I-hFSH uptake was affected by variations in ionic strength, with a maximum uptake, 2.17 x 10-16 moles per mg of homogenate, being noted in 1 mm phosphate buffer, pH 7.5 and no specific uptake in 0.5 m phosphate buffer, Specific uptake was not affected by substitution of Mg2+ for Ca2+ at the molar concentrations of phosphate described above. Specific binding is temperature-dependent and maximum specific uptake of 125I-hFSH was seen at 37°. Heating of seminiferous tubule homogenate to 60° and above prior to addition of 125I-hFSH abolished specific uptake, whereas nonspecific uptake increased as the temperature was elevated. Specific binding was pH-dependent, with an optimum at pH 7.5. Extremes of pH caused irreversible damage to the receptors and this was a time-dependent process. In our system, specific uptake of 125I-hFSH was a saturable process with respect to hormone concentration, and significant inhibition of uptake by unlabeled hFSH could be detected at 1.06 x 10-16 moles per mg of homogenate using 7.5 x 10-14 moles of 125I-hFSH. Scatchard analysis indicated a Kd of 6.7 x 10-10 m and binding sites are estimated to the 6.2 x 10-14 moles per mg wet tissue weight.

Ovine and human follicle-stimulating hormone (FSH) were modified by periodate oxidation followed by borohydride reduction, yielding FSH-containing analogues of N-acetylneuraminic acid (NANA) shortened by 1 or 2 carbon atoms. The FSH activity of the modified preparations was assayed by the human chorionic gonadotropin augmentation assay. The treatment decreased the biological activity of FSH somewhat, but, even when no unmodified NANA remained, the potency was still about half that of untreated FSH. Treatment of either native or modified FSH with neuraminidase resulted in the release of all NANA and NANA-8 and some NANA-7, and the loss of hormonal activity. It appears, therefore, that an intact 3-carbon polyhydroxy side chain in NANA is not required for full FSH activity.