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.

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).

High-affinity folate binding in human mammary gland

1993 ◽  
Vol 13 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Mammary Gland ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Gel Chromatography ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Mammary Gland Tissue ◽  
Human Mammary Gland ◽  
Triton X

High-affinity 3H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (Mr ≈ 100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.

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.

Characterization of a High Affinity Folate Binding Protein in Porcine Serum: Ionic Charge, Concentration—Dependent Polymerization and Ligand Binding Mechanism

2003 ◽  
Vol 23 (5-6) ◽  
pp. 339-351 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Affinity Chromatography ◽  
Binding Protein ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Exchange Chromatography ◽  
Ionic Charge ◽  
Folate Binding Protein ◽  
Porcine Serum ◽  
Hill Coefficients ◽  
Triton X

The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-af.nity type with upward convex Scatchard plots and Hill coefficients >1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/1. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing heterodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.

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).

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

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.

scholarly journals High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein

1991 ◽  
Vol 280 (1) ◽  
pp. 267-271 ◽  
Author(s):  
J Holm ◽  
S I Hansen ◽  
M Høier-Madsen ◽  
L Bostad
Keyword(s):  
Human Milk ◽  
Molecular Mass ◽  
Choroid Plexus ◽  
Binding Protein ◽  
Marker Enzyme ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Human Choroid Plexus ◽  
Molecular Masses ◽  
Triton X

High-affinity [3H]folate binding in solubilized human choroid plexus homogenate displayed characteristics, e.g. apparent positive co-operativity, which are typical of specific folate binding. The highest folate-binding activity per g of protein was associated with the 27000 g membrane pellet where the membrane-marker enzyme gamma-glutamyltransferase had its main localization. Ultrogel AcA 44 chromatography revealed two major folate-binding proteins (molecular masses greater than 110 kDa and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) in the Triton X-100-solubilized membrane pellet. After exposure of the membrane pellet to phosphatidylinositol-specific phospholipase C there was only one large 25 kDa peak of folate binding. This could suggest that the folate-binding protein is anchored to the membrane by a glycosylphosphatidylinositol moiety, which can be inserted into Triton X-100 micelles and thus can give rise to forms of large molecular size on gel filtration. This notion was supported by the identical molecular masses of the greater than 110 kDa and 25 kDa folate-binding peaks determined by SDS/PAGE and immunoblotting. The folate-binding protein in choroid plexus cross-reacted with rabbit antibodies against the 25 kDa human milk folate-binding protein, and paraffin-embedded sections of choroid plexus showed immunostaining after exposure to rabbit anti-(human milk folate-binding protein) serum (1:8000 dilution).

Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C

1992 ◽  
Vol 12 (2) ◽  
pp. 87-93 ◽  
Author(s):  
Steen Ingemann Hansen ◽  
Jan Holm
Keyword(s):  
Human Milk ◽  
Choroid Plexus ◽  
Phospholipase C ◽  
Molecular Species ◽  
Binding Protein ◽  
Gel Filtration ◽  
Molecular Size ◽  
Membrane Anchor ◽  
Folate Binding Protein ◽  
Triton X

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosly phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.

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