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).

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.

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.

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).

scholarly journals Expression of the genes encoding CCAAT-enhancer binding protein isoforms in the mouse mammary gland during lactation and involution

1998 ◽  
Vol 334 (1) ◽  
pp. 205-210 ◽  
Author(s):  
Georgios SABATAKOS ◽  
Gareth E. DAVIES ◽  
Maria GROSSE ◽  
Anthony CRYER ◽  
Dipak P. RAMJI
Keyword(s):  
Transcription Factors ◽  
Mammary Gland ◽  
Dna Binding ◽  
Binding Protein ◽  
Mouse Mammary Gland ◽  
Binding Activity ◽  
Protein Isoforms ◽  
Dna Binding Activity ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the activation of gene expression in the mammary gland during lactation. We have therefore investigated the detailed expression profile of the C/EBP family during lactation and involution of the mouse mammary gland. The expression of C/EBPβ and C/EBPδ mRNA was low during lactation, increased dramatically at the beginning of involution and remained constant thereafter. In contrast, C/EBPα mRNA expression was relatively high during the early stages of lactation, declined to low levels during the late stages of lactation and at the start of involution, and increased again during involution. Electrophoretic mobility-shift assays showed a close correlation between the expression of the C/EBP genes and the functional C/EBP DNA-binding activity and, additionally, demonstrated the participation of heterodimers, formed from among the three proteins, in DNA–protein interactions. The DNA-binding activity of the activator protein 1 (AP1) family of transcription factors was also induced during involution. These results therefore point to potentially important regulatory roles for both the C/EBP and the AP1 family during lactation and involution of the mammary gland.

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).

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