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

1989 ◽  
Vol 9 (1) ◽  
pp. 93-97 ◽  
Author(s):  
Steen Ingemann Hansen ◽  
Jan Holm ◽  
Mimi Høier-Madsen
Keyword(s):  
Human Urine ◽  
Radioligand Binding ◽  
Binding Protein ◽  
Molecular Size ◽  
Enzyme Linked Immunosorbent Assay ◽  
Folate Binding Protein ◽  
Binding Characteristics ◽  
High Affinity ◽  
Large Peak ◽  
Apparently Healthy

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 (Mr∼25,000).

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

1990 ◽  
Vol 10 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Amniotic Fluid ◽  
Radioligand Binding ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Amniotic Fluid ◽  
Folate Binding Protein ◽  
Binding Characteristics ◽  
High Affinity

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 (Mr~25 000) and a minor one (Mr~100 000).

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics, ionic charge and molecular size

1985 ◽  
Vol 5 (8) ◽  
pp. 683-688 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
J⊘rgen Lyngbye
Keyword(s):  
Binding Protein ◽  
Molecular Size ◽  
Binding Activity ◽  
Ionic Charge ◽  
Folate Binding Protein ◽  
Human Leukocytes ◽  
Binding Characteristics ◽  
High Affinity ◽  
Chromatographic Profile ◽  
Triton X

High-affinity binding of [3H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (Mr approx. 25 000) in the front effluent and a smaller more acidic peak (Mr approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (Mr approx 25 000), typically 5–10% of the total binding activity.

Ligand Binding Characteristics of Two Molecular Forms, One Equipped with a Hydrophobic Glycosyl Phosphatidylinositol Tail, of the Folate Binding Protein Purified from Human Milk

2002 ◽  
Vol 22 (3-4) ◽  
pp. 455-463 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Ligand Binding ◽  
Human Milk ◽  
Radioligand Binding ◽  
Binding Protein ◽  
Folate Receptor ◽  
Molecular Size ◽  
Molecular Forms ◽  
Folate Binding Protein ◽  
Binding Characteristics ◽  
Scatchard Plots

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [3H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.

High-affinity folate binding in human prostate

1993 ◽  
Vol 13 (2) ◽  
pp. 99-105 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Human Milk ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Human Prostate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gel Chromatography ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Sds Page ◽  
Triton X

Binding of 3H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography reveled two major folate binding proteins (Mr≈100 and 25kDa), but only one single band (Mr ≈ 65–70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum 3H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n = 6).

Characterization of a High-Affinity Folate Receptor in Normal and Malignant Human Testicular Tissue

1999 ◽  
Vol 19 (6) ◽  
pp. 571-580 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen ◽  
Thomas Broe Christensen ◽  
Carl W. Nichols
Keyword(s):  
Human Milk ◽  
G Protein ◽  
Radioligand Binding ◽  
Binding Protein ◽  
Folate Receptor ◽  
Testicular Tissue ◽  
Hydrophobic Membrane ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Triton X

We have characterized the folate receptor in normal and malignant tissue from male gonads. Radioligand binding displayed characteristics typical of other folate receptors. Those included a high-affinity type of binding (K = 1010 M−1), apparent positive cooperativity changing into non-cooperativity at low receptor concentrations, a tendency to increased binding affinity with decreasing receptor concentrations, a slow dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition by folates, in particular oxidized forms. The gel filtration profile of Triton X-100 solubilized tissue contained a 25 and 100 kDa peak of radioligand-receptor. The latter peak could represent receptor equipped with a hydrophobic membrane anchor that inserts into Triton X-100 micelles. The concentration of radiolabelled receptor ranged from 0.41 nmol/g protein to 1.68 nmol/g protein in specimens of normal testicular tissue from patients with prostatic carcinomas and from 1.54 nmol/g protein to 3.82 nmol/g protein in testicular tissue from young individuals. Compared to normal testicular tissue the concentration of receptor in seminoma tissue was low (0.38–1.27 nmol/g protein) but showed a higher degree of immunoreactivity in the presence of antibodies against human milk folate binding protein as evidenced by ELISA and immunohistochemistry data. Hence a folate receptor isoform homologous to human milk folate binding protein is apparently expressed in seminomas where the total expression of receptor, however, seems to be lower than in normal testicles.

A high-affinity folate binding protein in human semen

1991 ◽  
Vol 11 (5) ◽  
pp. 237-242 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Semen ◽  
Folate Binding Protein ◽  
High Affinity ◽  
A Minor ◽  
Two Peaks

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.

Binding Characteristics of Folate to High Affinity Folate Binding Protein Purified from Porcine Serum.

1999 ◽  
Vol 61 (7) ◽  
pp. 743-748 ◽  
Author(s):  
Masahiro NATSUHORI ◽  
Maki OKADA ◽  
Ryo IDA ◽  
Kazuaki SASAKI ◽  
Minoru SHIMODA ◽  
...  
Keyword(s):  
Binding Protein ◽  
Folate Binding Protein ◽  
Binding Characteristics ◽  
Porcine Serum ◽  
High Affinity

Foetal steroid binding protein in British and Japanese women

1987 ◽  
Vol 114 (4) ◽  
pp. 584-588 ◽  
Author(s):  
M. Jawed Iqbal ◽  
Alastair Forbes ◽  
Mark L. Wilkinson ◽  
John W. Moore ◽  
Roger Williams ◽  
...  
Keyword(s):  
Ovarian Function ◽  
Binding Protein ◽  
Premenopausal Women ◽  
Japanese Women ◽  
Sex Hormone Binding Globulin ◽  
Partial Purification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Characteristics ◽  
Steroid Binding ◽  
Steroid Binding Protein

Abstract. In order to examine the newly-discovered sex-steroid binding protein, foetal steroid binding protein (FSBP) in different populations, its binding characteristics and its level were studied by two-tier column ligand binding assay and enzyme-linked immunosorbent assay (ELISA) respectively. In 10 Japanese premenopausal women, analysis of 5α-dihydrotestosterone (DHT) binding in the Cibacron Blue 3GA-Sepharose 6B portion of the column showed a rising plateau pattern with a mean maximum binding of 31.1 ± 7.41%, whereas of 9 similar British women, 8 displayed unsaturable, non-cooperative binding of 11.6 ± 8.22% (P < 0.01). After partial purification of FSBP in these samples, the protein exhibited saturable binding kinetics, median binding 25 (interquartiles 23–34) and 19 (13–25) nmol DHT/l in Japanese and British women, respectively (P < 0.05). By analyzing FSBP by ELISA in 56 Japanese (45 premenopausal) and 59 British (25 premenopausal) women, higher levels were obtained in the whole Japanese group (P = 0.0016) and in the premenopausal Japanese women (P = 0.018) than in their British counterparts. In both nationalities, FSBP levels were higher in premenopausal women, and there was a significant negative correlation of FSBP with age in both populations, particularly in postmenopausal women. FSBP levels did not correlate with weight, parity, sex hormone binding globulin or albumin levels. The influence of FSBP on free steroid levels remains unclear, but some relationship with ovarian function seems a possibility.

scholarly journals Molecular target sizes of inositol 1,4,5-trisphosphate receptors in liver and cerebellum

1990 ◽  
Vol 265 (2) ◽  
pp. 393-398 ◽  
Author(s):  
D L Nunn ◽  
B V L Potter ◽  
C W Taylor
Keyword(s):  
Binding Sites ◽  
Radioligand Binding ◽  
Inositol Phosphate ◽  
Rank Order ◽  
Binding Protein ◽  
Molecular Target ◽  
Binding Studies ◽  
Single Class ◽  
Surface Receptors ◽  
High Affinity

Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.

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