scholarly journals Affinity labelling of the folate-binding protein in pig intestine

1990 ◽  
Vol 267 (1) ◽  
pp. 249-252 ◽  
Author(s):  
A M Reisenauer
Keyword(s):  
Folic Acid ◽  
Brush Border ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
Vacuum Filtration ◽  
Affinity Labelling ◽  
Hydroxysuccinimide Esters ◽  
Affinity Reagent ◽  
Filtration Technique ◽  
Specific Transport System

A specific transport system for folate and a high-affinity folate-binding protein have been identified in pig intestinal brush-border membranes. To determine if the binding protein plays a role in folic acid (PteGlu) uptake in to the cell, the inactivation of folate binding and transport by N-hydroxysuccinimide esters of folic acid (NHS-PteGlu) was compared. In addition, the number of brush-border proteins modified by the affinity reagent was assessed. Brush-border vesicles were incubated with various concentrations of NHS-PteGlu or NHS-methotrexate. Transport and binding of [3H]PteGlu by the vesicles were measured at 37 and 4 degrees C respectively by using the vacuum-filtration technique. NHS-methotrexate and NHS-PteGlu specifically inhibited PteGlu transport. Incubating the vesicles with 1 microM-NHS-PteGlu inactivated [3H]PteGlu transport by 60% and binding by 80%. Half-maximal inhibition of both transport and binding was observed at similar concentrations of the affinity reagent (0.05 and 0.07 microM-NHS-PteGlu respectively). Treating the vesicles with radiolabelled NHS-PteGlu followed by gel electrophoresis and autoradiography revealed a specifically labelled protein with an Mr of 56,000. These results indicate that the intestinal folate-binding and transport proteins are identical and that the function of the folate-binding protein is to transport folate into the cell.

Renal folate absorption and the kidney folate binding protein. II. Microinfusion studies

1987 ◽  
Vol 252 (4) ◽  
pp. F757-F760 ◽  
Author(s):  
J. Selhub ◽  
S. Nakamura ◽  
F. A. Carone
Keyword(s):  
Folic Acid ◽  
Brush Border ◽  
Half Life ◽  
Binding Protein ◽  
Membrane Surface ◽  
Rapid Change ◽  
Acid Uptake ◽  
Folate Binding Protein ◽  
Acid Absorption ◽  
Folic Acid Absorption

Surface proximal convoluted tubules (PCT) in rats were microinfused in situ with [3H]folic acid to study the role of folate binding protein (FBP) in the kidney brush-border membrane for renal conservation and transport of folate [3H]folic acid absorption was linearly related to tubular length of PCT and occurred largely in this segment of the tubule. Unlabeled folate derivatives inhibited [3H]folic acid absorption, the extent of which was dependent on the type of unlabeled folate used and its concentration. At equivalent concentrations, inhibition was most effective with unlabeled folic acid, slightly lower than with 5-methyltetrahydrofolate and least effective with methotrexate. Comparisons between [3H]folic acid absorption before and after infusion of a saturating dose of unlabeled folic acid or repetitive injections of [3H]folic acid into the same tubular site revealed continuous and rapid regeneration of unsaturated folic acid uptake sites with an apparent half-life of 28.75 +/- 8.75 s. Determination of [3H] retained in the tubule at various periods after microinfusion of [3H]folic acid revealed slow cellular disappearance with an apparent half-life of 47.3 +/- 5.4 min. It is proposed that the brush-border FBP functions as a receptor of infused folic acid and that following the binding of the ligand the folic acid/FBP complex undergoes a rapid change that results in the internalization of folic acid and regeneration of unsaturated binding sites at the membrane surface. Internalized folic acid is slowly released into renal capillaries.

Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule

1993 ◽  
Vol 264 (2) ◽  
pp. C302-C310 ◽  
Author(s):  
H. Birn ◽  
J. Selhub ◽  
E. I. Christensen
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Brush Border ◽  
Intracellular Transport ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Binding Protein ◽  
Rat Kidney ◽  
Folate Binding Protein

Folate-binding protein (FBP) is involved in folate reabsorption in the renal proximal tubule. Immunocytochemical studies have located FBP to the brush-border membrane, endocytic vacuoles, and dense apical tubules. We applied the same polyclonal antibody (anti-FBP) against FBP to investigate the dynamic relationship between FBP in the different compartments by microinjecting the antibody into rat kidney proximal tubules in situ. Specific binding of anti-FBP in vivo to the brush-border membrane was followed by fixation at various times. Protein A-gold labeling shows that anti-FBP is transported from endocytic invaginations into vacuoles followed by transport into dense apical tubules within 15 s. Thus FBP is rapidly internalized, and together with previous studies this study strongly suggests recycling of FBP back to the luminal plasma membrane through dense apical tubules. The results are consistent with reabsorption of folate through endocytosis of the FBP-folate complex followed by dissociation and recycling of FBP. When time is allowed there is a steady accumulation of FBP in dense apical tubules combined with an increase in surface density of the same compartment. A possible explanation involves partial inhibition of the fusion between dense apical tubules and plasma membrane because of the anti-FBP labeling of the receptor.

scholarly journals Folic acid and folate binding protein in pregnancy.

1977 ◽  
Vol 23 (5) ◽  
pp. 447-453 ◽  
Author(s):  
Suvit AREEKUL ◽  
Petcharin YAMARAT ◽  
Manit VONGYUTHITHUM
Keyword(s):  
Folic Acid ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
In Pregnancy

scholarly journals Influence of goat's-milk folate-binding protein on transport of 5-methyltetrahydrofolate in neonatal-goat small intestinal brush-border-membrane vesicles

1988 ◽  
Vol 59 (3) ◽  
pp. 497-507 ◽  
Author(s):  
Dallynn. Salter ◽  
Peter Blakeborough
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Molar Ratio ◽  
Folate Binding Protein ◽  
Intestinal Brush Border ◽  
Goat’S Milk ◽  
Goat's Milk

1. The influence of goat's-milk folate-binding protein (FBP) on the uptake of 5-methyltetrahydrofolate (MTHF) by brush-border-membrane vesicles prepared from the small intestine of the 6-d-old goat was investigated using a rapid-filtration assay.2. Uptake of MTHF by the membrane vesicles was strongly enhanced by FBP within the pH range 4·5-6·5, with an optimum at pH 5-5·5.3. Both the initial rate of MTHF uptake and uptake of MTHF at equilibrium were markedly increased in the presence of FBP.4. Uptake of MTHF by brush-border-membrane vesicles was maximal when the molar ratio FBP: MTHF was 1·0-2·5.5. The relation between pH and 125I-labelled FBP binding to the membranes was similar to that for uptake of MTHF, with an optimum at pH 5.6. In experiments in which the osmotic pressure of the incubation medium was progressively increased with cellobiose, 125I-labelled FBP was found to be taken up primarily by binding to the brush-border-membrane surface.7. Uptake of 125I-labelled FBP was time-dependent and saturable, with a Km of 0·39 (SE 0·07) μM and Vmax of 6·73 (SE 0·92) μg/mg protein.8. Experiments in which various milk proteins (cow FBP, goat FBP, α-lactalbumin, β-lactoglobulin, bovine serum albumin and lactoferrin) were allowed to compete in turn with 125I-labelled FBP for uptake by brush-border-membrane vesicles indicated that high-affinity binding was probably specific to FBP, although lactoferrin reduced uptake possibly by non-specific coating of the mucosal surface.9. It was concluded that a folate transport mechanism mediated by the FBP in milk exists at the intestinal brush border of neonatal goats. It is suggested that this may reinforce the developing endogenous transport system.

Cell fractionation and electron microscope studies of kidney folate-binding protein

1991 ◽  
Vol 260 (2) ◽  
pp. C338-C346 ◽  
Author(s):  
J. T. Hjelle ◽  
E. I. Christensen ◽  
F. A. Carone ◽  
J. Selhub
Keyword(s):  
Electron Microscope ◽  
Proximal Tubule ◽  
Brush Border ◽  
Binding Protein ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Cell Fractionation ◽  
Folate Binding Protein ◽  
Em Autoradiography ◽  
Endocytic Vesicles

The subcellular distribution of folate-binding protein (FBP) and [3H]folate in the proximal tubule was examined using cell fractionation and different electron microscope (EM) techniques. Cell fractionation of rabbit proximal tubules revealed that FBP distributed into two modes: 50% of FBP distributed with alanylaminopeptidase activity (brush border), and the remaining FBP distributed with organelles of lower density that did not show a large digitonin-induced shift to greater density. Infusion of [3H]folate into the kidney followed by isolation and fractionation of the proximal tubules revealed a time-dependent shift of [3H]folate from the heavy (brush border) mode to the lighter organelle mode. By EM immunocytochemistry, rat kidney FBP locates in the brush border, endocytic invaginations, endocytic vacuoles, and dense apical tubules of proximal tubule cells. EM autoradiography of rat kidney 10 min after intravenous infusion of [3H]folate revealed that the label was significantly concentrated only in the brush border, endocytic vesicles, and lysosomes. These data support a mechanism of receptor-mediated endocytosis for the process of FBP-mediated folate transport in the kidney.

Folate binding protein: therapeutic natural nanotechnology for folic acid, methotrexate, and leucovorin

Nanoscale ◽  
2017 ◽  
Vol 9 (7) ◽  
pp. 2603-2615 ◽  
Author(s):  
Rachel L. Merzel ◽  
Sarah M. Boutom ◽  
Junjie Chen ◽  
Carolina Frey ◽  
Kerby Shedden ◽  
...  
Keyword(s):  
Folic Acid ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
Protein Therapeutic

Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor

Biochemistry ◽  
1992 ◽  
Vol 31 (12) ◽  
pp. 3236-3243 ◽  
Author(s):  
He Chung Lee ◽  
Ryosuke Shoda ◽  
Jennifer A. Krall ◽  
John D. Foster ◽  
Jacob Selhub ◽  
...  
Keyword(s):  
Brush Border ◽  
Binding Protein ◽  
Folate Binding Protein ◽  
Brush Border Membranes

scholarly journals The heterogeneity and properties of folate binding proteins from chronic myelogenous leukemia cells

Blood ◽  
1975 ◽  
Vol 46 (6) ◽  
pp. 855-867 ◽  
Author(s):  
CD Fischer ◽  
M da Costa ◽  
SP Rothenberg
Keyword(s):  
Molecular Weight ◽  
Folic Acid ◽  
Chronic Myelogenous Leukemia ◽  
Binding Proteins ◽  
Binding Protein ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Folate Binding Protein ◽  
Binding Factor ◽  
Chronic Myelogenous Leukemia Cells

Previous studies have demonstrated that some chronic myelogenous leukemia cells contain a macromolecular binding factor for folic acid. This binder, which previously was believed to be a single factor, has now been resolved into two distinct binding proteins. Separation of each binder was obtained by DEAE chromatography of the partially purified lysate of chronic myelogenous leukemia cells. One binder has a molecular weight of 30;000–35,000, and the second binder has a molecular weight of 40,000-45,000. Both proteins bind the mono-, di-, and triglutamates of folic acid, N10-methyl-folate, dihydro-folate, and N5-methyltetrahydrofolate. Neither binder has determinants for N5- formyltetrahydrofolate or methotrexate. The preferred substrates for both binders appear to be the fully oxidized and partially reduced folates rather than the fully reduced folates. The lower-molecular- weight folate binding protein shows reversible binding with partially and fully reduced folates but irreversible binding with oxidized folates. This property suggests that this binder may have some function in the transport and storage of folate. The higher-molecular-weight folate binding protein, however, has only slight reversibility of binding with the partially and fully reduced folates, and it is therefore more difficult to postulate a physiologic function for this binding factor.

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