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.

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.

scholarly journals Isolation of membrane-bound renal enzymes that metabolize kinins and angiotensins

1976 ◽  
Vol 157 (3) ◽  
pp. 643-650 ◽  
Author(s):  
P E Ward ◽  
E G Erdös ◽  
C D Gedney ◽  
R M Dowben ◽  
R C Reynolds
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Surface Membrane ◽  
Renal Kallikrein ◽  
Proximal Tubular ◽  
Kallikrein Activity

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures. Kallikrein was highly concentrated in a plasma-membrane fraction but was absent from the brush-border membrane of proximal tubular cells. Cells of transplanted renal tumours of the rat, originating from the proximal tubule, had no kallikrein activity. Kininase activity, angiotensin I-converting enzyme (kininase II) and angiotensinase were found in a plasma-membrane-enriched fraction and especially in the fraction containing isolated brush border. It is suggested that after renal kallikrein is synthesized on endoplasmic reticulum, it is subsequently reoriented to a surface membrane for activation and release. Renal kallikrein may enter the tubular filtrate distal to the proximal tubules. The brush-border membrane of proximal tubule is the major site of inactivation of kinins and angiotensin II..

Immunolocalization of NHE8 in rat kidney

2005 ◽  
Vol 288 (3) ◽  
pp. F530-F538 ◽  
Author(s):  
Sunita Goyal ◽  
SueAnn Mentone ◽  
Peter S. Aronson
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Surface Membrane ◽  
Proximal Tubules ◽  
Intense Staining ◽  
Proximal Tubule Cells ◽  
Nephron Segment ◽  
Tubule Cells

In situ hybridization studies demonstrated that Na+/H+ exchanger NHE8 is expressed in kidney proximal tubules. Although membrane fractionation studies suggested apical brush-border localization, precise membrane localization could not be definitively established. The goal of the present study was to develop isoform-specific NHE8 antibodies as a tool to directly establish the localization of NHE8 protein in the kidney by immunocytochemistry. Toward this goal, two sets of antibodies that label different NHE8 epitopes were developed. Monoclonal antibody 7A11 and polyclonal antibody Rab65 both specifically labeled NHE8 by Western blotting as well as by immunofluorescence microscopy. The immunolocalization pattern in the kidney seen with both antibodies was the same, thereby validating NHE8 specificity. In particular, NHE8 expression was observed on the apical brush-border membrane of all proximal tubules from S1 to S3. The most intense staining was evident in proximal tubules in the deeper cortex and medulla with a significant but somewhat weaker staining in superficial proximal tubules. Colocalization studies with γ-glutamyltranspeptidase and megalin indicated expression of NHE8 on both the microvillar surface membrane and the coated-pit region of proximal tubule cells, suggesting that NHE8 may be subject to endocytic retrieval and recycling. Although colocalizing in the proximal tubule with NHE3, no significant alteration in NHE8 protein expression was evident in NHE3-null mice. We conclude that NHE8 is expressed on the apical brush-border membrane of proximal tubule cells, where it may play a role in mediating or regulating ion transport in this nephron segment.

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.

Critical domains in the specific binding of radiolabelled Vip3Af insecticidal protein to brush border membrane vesicles from Spodoptera spp. and cultured insect cells

2021 ◽  
Author(s):  
Yudong Quan ◽  
Maria Lázaro-Berenguer ◽  
Patricia Hernández-Martínez ◽  
Juan Ferré
Keyword(s):  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membrane ◽  
Ex Vivo ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Cry Proteins ◽  
Competition Binding

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis ). In this study, we have set up the conditions to analyze the specific binding of 125 I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but that Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine-mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125 I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding, and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125 I-DI-III. In addition, the truncated protein 125 I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo. IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for the ex vivo toxicity to Sf21 cells.

Characterization and distribution of albumin binding protein in normal rat kidney

1996 ◽  
Vol 271 (1) ◽  
pp. F101-F107 ◽  
Author(s):  
A. L. Cessac-Guillemet ◽  
F. Mounier ◽  
C. Borot ◽  
H. Bakala ◽  
M. Perichon ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Albumin Binding ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Proximal Tubular

The mechanism by which proteins that pass through the glomerular basal lamina are taken up by proximal tubule cells is incompletely characterized. Past work has identified the kinetics of albumin binding to renal brush-border membrane. We have now purified and characterized albumin binding protein (ABP) and shown its distribution in renal proximal tubular cells. ABP was purified from rat renal proximal tubular cell brush-border membrane by affinity chromatography with rat serum albumin-Sepharose. The resulting ABP had two apparent molecular masses (55 and 31 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies to ABP were raised in rabbits and checked by immunoassay and immunoblotting. Light-microscopic immunohistochemistry showed ABP all along the proximal tubule in the pars convoluta and pars recta. Electron-microscopic immunohistochemistry showed labeling on microvilli and in apical endocytic vacuoles, dense apical tubules, and lysosomes. These results indicate that ABP is involved in proximal tubule endocytosis.

Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na+/H+ exchanger NHE3 at PKA consensus sites

2005 ◽  
Vol 289 (2) ◽  
pp. F249-F258 ◽  
Author(s):  
Hetal S. Kocinsky ◽  
Adriana C. C. Girardi ◽  
Daniel Biemesderfer ◽  
Thao Nguyen ◽  
SueAnn Mentone ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Subcellular Trafficking ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Type 3

Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na+/H+ exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.

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