Development of a specific radioimmunoassay for the placental folate receptor and related high-affinity folate binding proteins in human tissues

High-affinity membrane-associated and soluble folate binding proteins (FBPs) from human placenta, milk, and KB cells appear to share antigenic determinants [A. C. Antony et al. (1981) J. Biol. Chem. 256, 9684-9692 and (1985) 260, 14911-14917]. Iodination of a highly purified preparation of placental folate receptor (PFR) by various techniques resulted in significant denaturation of the PFR as evidenced by additional peaks of radioactivity on Sephacryl S-200 gel filtration in 1% Triton X-100. These denatured species had similar molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as radioiodinated and native PFR, and were also recognized, albeit with less efficiency, by specific rabbit antiserum raised against purified PFR. Since these denatured species failed to bind folate, they were specifically excluded from 125I-PFR by their inability to bind pteroylglutamate-Sepharose. This ws accomplished in a single step by iodination of PFR bound to the affinity column and elution of 125I-PFR under identical conditions that the native PFR was purified. The purified 125I-PFR comigrated with unlabeled PFR on SDS-PAGE and its elution profile on Sephacryl S-200 gel filtration was identical to radioligand bound PFR. The resulting radioimmunoassay standard curve using this affinity chromatography purified 125I-PFR, unlabeled PFR, and anti-human PFR serum had a range for measurement between 5 and 500 ng of PFR and was not affected by the concentration of folate in the sample. The practical utility of this radioimmunoassay for measuring cross-reacting material to the PFR was validated by its ability to quantitate the 40,000 and 160,000 Mr FBPs which are the two major forms of high-affinity FBPs in human tissues.     

Human placental coated vesicles contain receptor-bound transferrin.

Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation.     

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10¹⁰M^–1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 speciments of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.     

Rat liver nuclei contain receptors for a folate binding protein

A folate binding protein purified from the cytoplasm of human chronic myelogenous leukemia cells and saturated with [3H]pteroylglutamic acid, and the same protein labeled with 125I and saturated with pteroylglutamic acid, binds to the nuclear fraction of rat liver. EDTA inhibits this binding and this inhibition is reversed by Ca2+ but not by Mg2+. The nuclear fraction binds very little free [3H]pteroylglutamic acid, and the cytoplasm from which the nuclei have been removed does not bind the protein-folate complex. A Kd of 0.7 nM and a value of 1000 unsaturated binding sites per nucleus were obtained by Scatchard analysis. The translocation of folate to the nuclear membrane or nucleus by this soluble cytoplasmic folate binder may be the mechanism for the induction of enzyme(s) required for the metabolism of the folate ligand attached to the protein.     

Specific, high-affinity binding sites for angiotensin II on Mycoplasma hyorhinis.

Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.     

Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made.     

Avian species-specific tandem repeats contain nuclear protein binding sites.

Three avian highly repetitive tandem repeats were identified and examined. These repeats had similar unit lengths (about 42 bp long) but completely different sequences each containing particular protein binding sites. Each of these repeats was found within only one of the five closely related genera studied.     

Structuring of H1 histone. Evidence of high-affinity binding sites for phosphate ions

Circular dichroism studies show that low concentrations of phosphate ions induce folding of the H1 histones. Sulfate and perchlorate anions have effects similar to phosphate indicating the presence on H1 histones of binding sites with high affinity for ions with tetrahedral geometry. In fact, the structuring efficiency of different ions, as determined by the midpoint value of the effect/concentration curve, is 0.05 M for NaCl, 0.005 M for NaClO4, 0.001 M for sodium phosphate and 0.0003 M for sodium sulfate on H1 histone from Chaetopterus variopedatus sperm chromatin. Phosphate shows similar folding efficiency also on calf thymus and on sea-urchin sperm H1 histones. The effect of phosphate ions on the H1 molecule is observed also by differential absorption spectroscopy in the region of absorption of amino acid side-chains. Binding studies by gel filtration chromatography on Sephadex columns show that phosphate binding occurs in the presence of structuring concentrations of sodium chloride. About 9 ATP molecules bind to H1 histones derived from non-active cell chromatins while only 3.5 ATP molecules bind to H1 derived from active somatic chromatins. The fluorescence of the tyrosine residues of Chaetopterus sperm H1 is enhanced by chloride ions and heavily quenched by phosphate ions in correlation with structuring of the molecule, demonstrating direct interactions between tyrosine residues and phosphate ions. The defined and limited number of phosphate groups bound per histone molecule, the high affinity of the interaction and the effect on the structure of the histone suggest the participation of phosphate groups in the binding of H1 histones to DNA.     

A high-affinity folate binding protein in human urine. Radioligand binding characteristics,immunological properties and molecular size

High-affinity binding of [³H]folate in human urine displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. By means of a two-site enzyme-linked immunosorbent assay (ELISA) with rabbit antibodies against the low molecular weight folate binding protein from human milk, we measured folate binding protein concentrations in the range of 0.51 to 4.13 nM in urine samples from 16 apparently healthy individuals. Ultrogel AcA 44 chromatography of the urine showed that immunoreactive and radioligand bound folate binding protein coeluted in one large peak (M_r25,000).     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Interactions of sterols with antiestrogen-binding sites: structural requirements for high-affinity binding

Animal and human tissues contain a microsomal protein that binds nonsteroidal antiestrogens with high affinity and specificity. The functions of these binding sites and the identity of their natural ligands are unknown. Following a report that certain sterols inhibit [3H]tamoxifen binding to this site, we attempted to define the structural requirements for maximal inhibition of [3H]tamoxifen binding to rat liver antiestrogen-binding sites. Our studies identified 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) as the most potent sterol, having an inhibitory activity that was 12% that of unlabeled tamoxifen and an equilibrium dissociation constant of 6.3 nM. Structural features that appeared important for the inhibitory activity of this sterol include the presence of i) a hydrocarbon side chain at C17; ii) an oxygen function at C7; iii) a hydroxyl group at C3; and iv) the absence of a double-bond between C5 and C6. Saturation analysis and kinetic studies of [3H]tamoxifen binding in the presence of varying concentrations of 7-ketocholestanol clearly indicated that this sterol competed directly with tamoxifen for the antiestrogen-binding site. Unlike tamoxifen, this sterol did not bind to the estrogen receptor. These features make 7-ketocholestanol a potentially valuable tool for studying the properties and functions of this site.     

Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain golgins, whose C termini bind the Arf-like 1 G protein on the trans-Golgi, can also bind four members of the Rab family of G proteins. The Rab2-, Rab6-, Rab19-, and Rab30-binding sites are within the coiled-coil regions that are not required for Golgi targeting. Binding sites for two of these Rabs are also present on two coiled-coil proteins of the cis-Golgi, the Drosophila melanogaster orthologues of GM130 and GMAP-210. We suggest an integrated model for a tentacular Golgi in which coiled-coil proteins surround the Golgi to capture and retain Rab-containing membranes, excluding other structures such as ribosomes. Binding sites for diverse Rabs could ensure that incoming carriers are captured on first contact and moved to their correct destination within the stack.     

Azidomorphine is an agonist of high-affinity opioid receptor binding sites

Azidomorphine at low concentration (10^–9 M) inhibits the high-affinity binding site of labeled naloxone in rat brain membrane preparations. In the presence of Na⁺ and guanine nucleotides the displacement curves of azidomorphine are increased toward high concentrations, whereas Mg²⁺ ions decrease the IC₅₀ values; This demonstrates the agonist behavior of azidomorphine in binding experiments. When compared with morphine, azidomorphine displayed five-fold lower IC₅₀ values. Based on the presented results, azidomorphine appears to be a good candidate for photoaffinity labeling of opiate receptors.     

Intestinal epithelial cells and musculature contain different muscarinic binding sites

Muscarinic receptors on epithelial cells mediate intestinal secretion, while those in intestinal smooth muscle mediate motility. Experiments were carried out to determine whether the muscarinic receptors mediating each of these two functions in intestinal tissue might be associated with differences in the way agonist and antagonist drugs interact with the receptors. The inhibition constant (Kj) values for atropine, pirenzepine, and oxotremorine competition of specifically bound (3H)QNB were determined using membrane preparations from the muscular coat and from epithelial cells of rat jejunum, ileum, and colon. The Kj values of atropine were similar (1.2-10 nM) when comparing muscle layers and epithelial cells from any intestinal region. In contrast, the Kj values for pirenzepine were significantly higher in membranes from the musculature (400-1,200 nM) than in any of the epithelial cell membranes (20-100 nM). Kj values for pirenzepine in gut muscle were similar to those in heart (300 nM), whereas the Kj values in the cerebral cortex (39 nM) and the epithelial cell membranes closely approximated one another. The Kj values for oxotremorine competition of QNB binding in all intestinal muscular tissues (29-48 nM) and in heart (16 nM) were less than those of the intestinal epithelial cells (100-1,300 nM) or cerebral cortex (71 nM). Thus, pirenzepine and oxotremorine binding studies show that the nature of interactions between these agents and muscarinic sites is different when comparing epithelial cells and musculature of the gut.     

External labelling of glycoproteins from first-trimester human placental microvilli.

The brush-border glycoproteins of first-trimester human placentas were investigated by using two external labelling techniques: (1) sequential digestion with neuraminidase and galactose oxidase, followed by reduction with NaB3H4, which 3H-labels terminal galactose and galactosamine residues; and (2) sequential treatment with periodate and NaB3H4, which 3H-labels terminal sialic acid residues. The labelling procedures were performed on intact tissue so that the results would more closely approximate the topography of the brush border in vivo. The microvilli were isolated, subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the [3H]glycoproteins detected by fluorography. Densitometer scans of the fluorograms of the [3H]galactoproteins showed that, under reducing conditions, 90% of the protein-associated radioactivity was incorporated into two glycoproteins. The major [3H]galactoprotein of early placental microvilli had an estimated molecular mass of 92 kDa (desialylated) and migrated as a diffuse band. A minor 180 kDa glycoprotein was less consistently labelled. No change in the apparent molecular mass of either component was detected in the absence of beta-mercaptoethanol, suggesting that the 180 kDa component was not a dimer of the 92 kDa glycoprotein. The remaining 10% the radioactivity was equally distributed among several minor membrane components. Densitometer scans of the fluorograms of the [3H]sialoproteins showed that, under either reducing or non-reducing conditions, 90% of the 3H was preferentially incorporated into the 92-110 kDa region of the gel. Although no distinct bands were visible, the higher-molecular-mass region of this area was always most heavily labelled. A minor 180 kDa glycoprotein was also 3H-labelled. The pattern of brushborder [3H]glycoproteins from first-trimester placentas differed markedly from that of term placental microvilli and from placental fibroblast plasma membranes that were 3H-labelled by identical external labelling techniques. These results indicate that: (1) the glycoprotein determinants of brush-border topography change during pregnancy; (2) within the placenta, the major 92 kDa (desialylated) determinant, which has not been previously described, is unique to the trophoblastic cells.     

Murine erythroleukaemia cells (Friend cells) possess high-affinity binding sites for erythropoietin

Murine erythroleukaemia cells represent erythroid precursors blocked near the CFU-E or proerythroblast stage. In contrast to their non-leukaemic equivalents, neither their proliferation nor their differentiation seems to be affected by erythropoietin. However, we show in this paper that both uncommitted and committed, benzidine-positive, cells bind iodinated erythropoietin. The binding is of high affinity (Kd = 490 +/- 160 pM) and reversible with a half-life of the complex of 77 +/- 19 min. The number of binding sites is low (300-600 per cell). In contrast the haematopoietic non-erythroid cell lines HL 60 and L 1210 and the myeloid-erythroid human cell line K 562 do not exhibit specific binding. If these binding sites represent true hormone receptors, their presence on a permanent cell line should facilitate erythropoietin receptor purification.     

B lymphocytes from hyporesponsive SJL mice contain aberrant nucleoside binding sites

C8-substituted guanine ribonucleosides activate B cells by a novel pathway that apparently is independent of GTP-binding proteins and protein kinase C. B lymphocytes from SJL mice are hyporesponsive to antigen-independent inductive signals transmitted by these nucleosides. In the current studies, the basis for this observation was explored. Responses of normal murine strains to these agents have been dissociated into antigen-independent (inductive) and antigen-dependent (differentiative) types by use of the 7,8-disubstituted guanine ribonucleosides. Dose-response profiles for inductive responses appear to correlate with apparent Kd values for low-affinity nucleoside binding sites; dose-response curves for antigen-dependent differentiative responses correlate with apparent Kd values for high-affinity binding sites. It was found that the SJL low-affinity site exhibits an apparent Kd that is approximately 10- to 20-fold lower in affinity for 8BrGuo than that of normal CBA mice. Although the low-affinity site in normal murine strains displays nearly equivalent affinity toward C8-substituted and 7,8-disubstituted nucleosides, the low-affinity site of SJL mice binds 7,8-disubstituted compounds with approximately 5-fold higher affinity than it does monosubstituted compounds. The dissociation constant for high-affinity nucleoside binding sites of SJL mice was only slightly different from that of CBA mice, consistent with the observation of essentially normal antigen-dependent nucleoside-mediated activity in SJL mice. The current observations support (a) a role for low-affinity binding sites in antigen-independent inductive events, (b) a role for high-affinity binding sites in antigen-dependent differentiative events mediated by substituted guanine nucleosides, and (c) the existence of aberrant low-affinity binding sites in B cells from SJL mice.     

Specific high-affinity binding sites for a synthetic gliadin heptapeptide on human peripheral blood lymphocytes

The synthetic peptide containing residues 43-49 of alpha-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. We now demonstrate using radiolabeled alpha-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (KD) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of alpha-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of [125I]alpha-gliadin(43-49) than T-lymphocytes. The lymphocyte alpha-gliadin(43-49) receptor may play an important role in mediating the immunological response to alpha-gliadin.     

Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen.

The efficiency of replication of plasmids containing the control region of polyomavirus DNA including one, two, or all three of the strong binding sites for large T antigen was measured in COP 8 cells which provide polyomavirus T antigen in trans. It was found that plasmids carrying only binding site A (the one closest to the origin core region) exhibited only 10% of the replication competence of plasmids with binding sites A and B or A and C. Plasmids containing all three binding sites, A, B, and C, did not replicate more efficiently than those with only two strong T-antigen-binding sites. We conclude, therefore, that optimal T-antigen-dependent replication of polyomavirus DNA requires two high-affinity T-antigen-binding sites.