The high-affinity folate binding protein in normal and malignant mammary gland tissue.
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Breast↗
Biomedical subjects
S I Hansen
Publications and source records attributed to S I Hansen.
High-affinity folate binding in human mammary gland.
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 (M(r) approximately 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%.
Breast↗
High-affinity folate binding in human prostate.
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 revealed two major folate binding proteins (M(r) approximately 100 and 25 kDa), but only one single band (M(r) approximately 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).
Carrier Proteins↗
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.
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 glycosyl phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.
Carrier Proteins↗
A high-affinity folate binding protein in proximal tubule cells of human kidney.
High-affinity 3H-folate binding in solubilized brush border membranes of human kidney cortex display characteristics such as apparent positive cooperativity typical of specific folate binding. The folate binding activity and the activity of the brush border membrane-marker enzyme, gamma-glutamyltransferase, were eightfold higher in brush border membranes compared to crude kidney homogenate. Ultrogel AcA 44 chromatography revealed a major (Mr approximately 100 kDa) and a minor (Mr approximately 25 kDa) folate binding protein in brush border membranes. The large molecular size form may represent a membrane-derived hydrophobic folate binding protein inserted in Triton X-100 micelles. This notion was supported by the identical molecular weights of the 100 kDa and 25 kDa folate binding peaks determined by sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotting. The folate binding protein in renal brush border membranes cross reacted with rabbit antibodies against 25 kDa human milk folate binding protein, and showed immunoprecipitation in the presence of these antibodies. Paraffin embedded sections of kidney cortex showed immunostaining of cells in the convoluted proximal tubules after exposure to rabbit antiserum (1:8000 dilution). Glomeruli and distal tubules showed no immunostaining.
Carrier Proteins↗
High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein.
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).
Binding Sites↗
A high-affinity folate binding protein in human semen.
The presence of a folate binding protein of high-affinity type (affinity constant 3.10(10) M-1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37 degrees C, pH 7.4) with the radioligand 3H-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 specimens 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.
Antibodies↗
High-affinity folate binding in human liver membranes.
High-affinity binding of 3H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel AcA 44 chromatography of solubilized membranes saturated with 3H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting.
Binding Sites↗
Conversion of an apparent 100 kDa folate binding protein from human milk to a 25 kDa molecular species by phospholipase D.
Gel filtration studies in the presence of Triton X-100 showed that treatment with phospholipase D reduced the apparent molecular size of the 100 kDa folate binding protein from human milk to 25 kDa, which is the molecular size of the low molecular weight folate binding protein. A phospholipase D induced cleavage of a hydrophobic phospholipid domain inserting the protein into Triton X-100 micelles could account for this phenomenon.
Carrier Proteins↗
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 5.10(9) M-1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37 degrees C, pH 7.4) with the radioligand 3H-folate. Dissociation of 3H-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 approximately 25,000) and a minor one (Mr approximately 100,000).
Amniotic Fluid↗
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 (Mr approximately 25,000).
Carrier Proteins↗
Serum antibodies to cow's milk folate-binding protein in patients with chronic inflammatory bowel disease.
Antibodies to folate-binding protein (FBP) of cow's milk were studied in serum from patients with chronic inflammatory bowel disease (CIBD), since earlier studies had shown IgG- and IgM-serum antibodies to other isotypes of milk proteins to be elevated in such patients, indicating a possible primary role or an epiphenomenon in the pathogenesis of CIBD. The amount of IgG antibodies to FBP was compared to disease activity, localization and duration by means of a newly developed enzyme-linked immunosorbent assay (ELISA). No statistically significant differences were seen between groups of patients with ulcerative colitis or Crohn's disease, as compared to healthy volunteers. Median values of FBP for ulcerative colitis were: 13 units/ml (4-74), for Crohn's disease: 14 units/ml (7-73), and healthy volunteers: 14 units/ml (4-51). No correlation was seen when comparing serum antibody values to disease activity, localization or duration. The intra-assay coefficient of variation of the ELISA technique was 0.05. The concentration of FBP in cow's milk (10 mg/l) is more than 50 times lower than that of other proteins (albumin, lactoglobulin, and casein). Even if a local defect in the endothelial barrier in these diseases may allow access of other milk protein antigens into the blood circulation, FBP is consequently not of importance for the humoral immune response in CIBD.
Adolescent↗
Quantification of biotin in serum by competition with solid-phase biotin for binding to peroxidase-avidin conjugate.
We have developed a simple, fast, accurate, and reproducible method--an Enzyme-Linked Ligand Sorbent Assay (ELLSA)--for quantifying biotin without the use of isotopes and immunological reagents. The analytical principle is based on the competition between biotin of the sample and immobilized biotin, i.e., biotinylated bovine serum albumin coated onto immunoplates, for binding to horseradish peroxidase (EC 1.11.1.7)-avidin conjugate. The amount of conjugate bound to immobilized biotin is subsequently measured.
Binding, Competitive↗
A competitive enzyme-linked ligand sorbent assay (ELLSA) for quantitation of folates.
An enzyme-linked ligand sorbent assay (ELLSA) for quantitation of folates is described. The method involves the following steps: (a) folate complexed to bovine serum albumin is adsorbed onto microtiter plates; (b) added folates compete with immobilized folate for binding to added biotinylated folate-binding protein; (c) biotinylated folate-binding protein bound to immobilized folate is detected after binding of avidin-alkaline phosphatase. The specificity of ELLSA is similar to that of conventional radioisotope dilution methods, and the sensitivity is high (lower limit of detection 20 fmol/sample). Quantitation of folates in erythrocyte lysates from 43 persons was performed by ELLSA. The results correlated fairly well with those obtained by the conventional radioisotope dilution method.
Alkaline Phosphatase↗
Rabbit antibodies against the low molecular weight folate binding protein from human milk. Use for immunological characterization of human folate binding proteins in an enzyme-linked immunosorbent assay (ELISA).
Antibodies to a low molecular weight folate binding protein isolated from human milk were raised in rabbits and used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for immunological characterization of human folate binding proteins (FBPs). The high and low molecular weight FBPs from human milk were immunologically indistinguishable. Furthermore, the FBPs in human urine and cerebrospinal fluid showed a cross-reactivity of 70% and 30%, respectively. No cross-reactivity of the FBP from cow's milk was observed.
Antibodies↗
Immobilized purified folate-binding protein: binding characteristics and use for quantifying folate in erythrocytes.
Purified folate-binding protein from cow's milk was immobilized on monodisperse polymer particles (Dynospheres) activated by rho-toluenesulfonyl chloride. Leakage from the spheres was less than 0.1%, and the binding properties were similar to those of the soluble protein with regard to dissociation, pH optimum for binding pteroylglutamic acid, and specificity for binding various folate derivatives. We used the immobilized folate-binding protein as binding protein in an isotope-dilution assay for quantifying folate in erythrocytes. The detection limit was 50 nmol/L and the CV over a six-month period was 2.3% (means = 1.25 mumol/L, n = 15). The reference interval, for folate measured in erythrocytes of 43 blood donors, was 0.4-1.5 mumol/L.
Carrier Proteins↗
Rabbit antibodies against the folate binding protein from cow's milk. Production, characterization and use for development of an enzyme-linked immunosorbent assay (ELISA).
Rabbit antibodies to purified folate binding protein from cow's milk whey were used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine folate binding proteins. The folate binding proteins in human milk and serum showed no cross-reactivity. A partial saturation of purified bovine folate binder with folate gave rise to an increased antigenicity probably due to a ligand (folate)-induced exposure of antigenic sites on the protein.
Animals↗
A high-affinity folate binding protein in normal human leukocytes: ligand binding characteristics, ionic charge and molecular size.
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 cell sediment from the leukocyte homogenate contained some high-affinity folate binding activity (Mr approx 25 000), typically 5-10% of the total binding activity.
Carrier Proteins↗