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.

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).

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 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.

Effect of Hydrogen Ion Concentration and Buffer Composition on Ligand Binding Characteristics and Polymerization of Cow's Milk Folate Binding Protein

2001 ◽  
Vol 21 (6) ◽  
pp. 745-753 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Ligand Binding ◽  
Binding Protein ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Cow’S Milk ◽  
Folate Binding Protein ◽  
Hydrogen Ion Concentration ◽  
Binding Characteristics ◽  
Cow's Milk ◽  
Buffer Composition

The ligand binding and aggregation behavior of cow's milk folate binding protein depends on hydrogen ion concentration and buffer composition. At pH 5.0, the protein polymerizes in Tris-HCl subsequent to ligand binding. No polymerization occurs in acetate, and binding is markedly weaker in acetate or citrate buffers as compared to Tris-HCl. Polymerization of ligand-bound protein was far more pronounced at pH 7.4 as compared to pH 5.0 regardless of buffer composition. Binding affinity increased with decreasing concentration of protein both at pH 7.4 and 5.0. At pH 5.0 this effect seemed to level off at a protein concentration of 10−6 M which is 100–1000 fold higher than at pH 7.4. The data can be interpreted in terms of complex models for ligand binding systems polymerizing both in the absence or presence of ligand (pH 7.4) as well as only subsequent to ligand binding (pH 5.0).

A Folate Binding Protein in Ascitic Fluid, Serum and Ovarian Tissue of Patients with Ovarian Adenocarcinoma Immunoreacts with Antibodies Against Human Milk Folate Binding Protein

1998 ◽  
Vol 18 (2) ◽  
pp. 49-57 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen ◽  
Poul-Erik Helkjær
Keyword(s):  
Human Milk ◽  
Ovarian Carcinoma ◽  
Ascitic Fluid ◽  
Radioligand Binding ◽  
Binding Protein ◽  
Ovarian Tissue ◽  
Tissue Homogenate ◽  
Ovarian Adenocarcinoma ◽  
Folate Binding Protein ◽  
Carcinoma Tissue

The presence of a folate binding protein which immunoreacts with antibodies against human milk folate binding protein was demonstrated in ascitic fluids from seven patients with ovarian adenocarcinoma. Ascitic fluids collected from two patients with other malignancies contained non-immunoreactive FBP. Tumor tissue specimens from five patients with ovarian carcinoma contained immunoreactive FBP. By contrast to normal ovaries ovarian carcinoma tissue showed positive immunostaining on immunohistochemistry. Ascitic fluids from two patients with ovarian carcinoma exhibited single distinct bands on SDS-PAGE immunoblotting. The gel filtration profile of ovarian carcinoma tissue homogenate from two patients contained 25 and 100 kDa peaks of radioligand-bound and immunoreactive folate binding protein, while ascitic fluid from one of the patients exhibited a large 100 kDa immunoreactive peak with no radioligand binding activity. The immunoreactive non-functional 100 kDa FBP could represent unprocessed precursor FBP. Future studies are necessary to evaluate whether determination of immunoreactive FBP in ovarian adenocarcinomatosis is of any diagnostic value.

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).

Ligand Binding and Polymerization Characteristics of Human Milk Folate Binding Protein Depend on Concentration of Purified Protein and Presence of Amphiphatic Substances

2003 ◽  
Vol 23 (2-3) ◽  
pp. 77-85 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Human Milk ◽  
Lipid Bilayers ◽  
Radioligand Binding ◽  
Gel Filtration ◽  
Hydrophobic Membrane ◽  
Folate Binding Protein ◽  
Binding Characteristics ◽  
Low Concentrations ◽  
Folate Binding Proteins ◽  
Triton X

Two folate binding proteins are present in human milk; one of 27 kDa is a cleavage product of the other one (100 kDa) which possesses a hydrophobic membrane anchor. A drastic change of radioligand binding characteristics and appearance of aggregated weak-radioligand affinity forms on gel filtration occurred at low concentrations of both proteins in the absence of Triton X-100 or other amphiphatic substances, e.g. cetyltrimethylammonium and phospholipids. These findings are consistent with a model predicting association between unliganded and liganded monomers resulting in weak-ligand affinity dimers. Amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers become hydrophilic in the liganded state) thereby preventing association between these monomeric forms prevailing at low concentrations of the protein. Bio-Gel P-300 chromatography of the 27 kDa protein revealed a pronounced polymerization tendency, which diminished with decreasing protein concentrations, however, not in the presence of cetyltrimethylammonium. The data could have some bearings on observations indicating that naturally occurring amphiphatic substances, cholesterol and phospholipids, are necessary for the important clustering of membrane folate receptors.

Sign in / Sign up

Export Citation Format

Share Document