scholarly journals Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes

1988 ◽  
Vol 255 (1) ◽  
pp. 185-191 ◽  
Author(s):  
H Debiec ◽  
R Lorenc
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
High Specificity ◽  
Brush Border Membranes ◽  
Intestinal Brush Border

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

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.

Isolation and function of a receptor for human lactoferrin in human fetal intestinal brush-border membranes

1991 ◽  
Vol 261 (5) ◽  
pp. G841-G846 ◽  
Author(s):  
H. Kawakami ◽  
B. Lonnerdal
Keyword(s):  
Human Milk ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Iron Absorption ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Constant ◽  
Binding Studies ◽  
Brush Border Membranes ◽  
Intestinal Brush Border

Iron absorption is known to be higher from human milk than from infant formula or bovine milk. The high bioavailability of human milk iron suggests that lactoferrin (Lf), the major iron-binding protein in human milk, may be a factor contributing to iron absorption in infants. We have isolated a human Lf receptor from solubilized human fetal intestinal brush-border membranes by affinity chromatography using immobilized human Lf. We also investigated the interaction of 125I-labeled human Lf and bovine Lf with brush-border membrane vesicles (BBMVs) from human small intestine using a rapid filtration technique. The molecular weight of the receptor was 110,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 37,000 under reducing conditions. Competitive binding studies demonstrated specific binding of human Lf. The binding was pH dependent, with an optimum between pH 6.5 and 7.5. Scatchard plot analysis indicated 4.3 x 10(14) binding sites/mg membrane protein with an affinity constant of 0.3 x 10(6) M-1 for human Lf. Both half-Lf and deglycosylated Lf bound to the receptor with an affinity similar to intact Lf. In contrast, little binding of bovine Lf or human transferrin to human BBMVs occurred. These results suggest that the brush-border membrane receptor for human Lf may be responsible for the high iron absorption from human milk.

scholarly journals Identification and characterization of the major stilbene-disulphonate- and concanavalin A-binding protein of the porcine renal brush-border membrane as aminopeptidase N

1990 ◽  
Vol 271 (1) ◽  
pp. 147-155 ◽  
Author(s):  
H See ◽  
R A F Reithmeier
Keyword(s):  
Molecular Mass ◽  
Concanavalin A ◽  
Brush Border ◽  
Membrane Fraction ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Aminopeptidase N ◽  
Brush Border Membranes ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

A 130 kDa glycoprotein (GP 130) was purified from porcine renal brush-border membranes by affinity chromatography using immobilized 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate (SITS)- and concanavalin A-Sepharose. GP 130 was the major concanavalin A-binding protein in porcine renal brush-border membranes and also bound Ricinus communis (castor-bean) and wheat-germ agglutinins. Endo-beta-N-acetylglucosaminidase F reduced the molecular mass of GP 130 by 20 kDa as determined by SDS/PAGE, whereas endo-beta-N-acetylglucosaminidase H reduced the molecular mass by 5 kDa, showing that GP 130 contained both complex and high-mannose carbohydrate structures. Western-blot analyses using an antibody raised against GP 130 showed that it was localized to the brush-border membrane fraction and was present in a membrane fraction of the pig kidney cell line LLC-PK1. The N-terminal sequence and amino acid composition of GP 130 showed that GP 130 is similar to rat kidney zinc peptidase and human intestinal aminopeptidase N. GP 130 had aminopeptidase N enzymic activity and was inhibited by bestatin (Ki = 36 microM), 1,10-phenanthroline (Ki 30 microM), Zn2+ (Ki 26 microM), Cu2+ (Ki 260 microM), pre-incubation with EDTA and by a polyclonal antibody against GP 130. Bicarbonate and iodide blocked the binding of GP 130 to the SITS-affinity resin, showing that GP 130 has an anion-binding site. Neither these anions nor stilbene disulphonates affected the aminopeptidase N activity of GP 130.

Reconstitution of intestinal Na(+)-phosphate cotransporter

1993 ◽  
Vol 264 (4) ◽  
pp. G609-G616 ◽  
Author(s):  
B. E. Peerce ◽  
M. Cedilote ◽  
S. Seifert ◽  
R. Levine ◽  
C. Kiesling ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Phosphate Uptake ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Preparative Scale ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border ◽  
Ph Range

The rabbit intestinal brush-border membrane Na(+)-phosphate cotransporter was purified from sodium dodecyl sulfate (SDS)-brush-border membrane vesicles (BBMV) protein (SDS-treated Ca(2+)-precipitated BBMV) by a three-column chromatography protocol. The purification included a preparative scale chromatofocusing chromatography column over the pH range from 7.4 to 4 after solubilization in 3-[(3-cholamidopropyl)-diamethylammonia]-1-propanesulfonate (CHAPS), a chromatofocusing column over the pH range from 5.6 to 4 after solubilization in n-octyl glucoside, and gel filtration chromatography on a Sephacryl S-200 column. Verification of Na(+)-phosphate cotransporter purification involved substrate affinities, substrate stoichiometry, and inhibitor sensitivity after proteoliposome reconstitution and SDS-polyacrylamide gel electrophoresis (PAGE). After gel filtration Na(+)-dependent phosphate uptake was 3,300-fold enriched compared with the cell homogenate. A single 130-kDa polypeptide was visualized by SDS-PAGE under reducing conditions using silver stain. The coenrichment of this 130-kDa polypeptide and proteoliposome reconstituted Na(+)-dependent phosphate uptake suggest that the intestinal brush-border membrane Na(+)-phosphate cotransporter has been purified and proteoliposome reconstituted.

Further characterization of a novel phospholipase B (phospholipase A2 – lysophospholipase) from intestinal brush-border membranes

1991 ◽  
Vol 69 (5-6) ◽  
pp. 346-357 ◽  
Author(s):  
Steven Pind ◽  
Arnis Kuksis
Keyword(s):  
Brush Border ◽  
Sodium Dodecyl Sulphate ◽  
Brush Border Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subcellular Fractionation ◽  
Polyclonal Antiserum ◽  
Brush Border Membranes ◽  
Intestinal Brush Border ◽  
Phospholipase B

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 – lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate – polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5–10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5–10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as a 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.Key words: phospholipase, brush-border membrane, intestine, phospholipid hydrolysis, antibodies.

Divalent cations and vitamin B12 uptake by intestinal brush-border membrane vesicles

1980 ◽  
Vol 239 (6) ◽  
pp. G452-G456
Author(s):  
R. C. Beesley ◽  
C. D. Bacheller
Keyword(s):  
Vitamin B12 ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Divalent Cations ◽  
Intrinsic Factor ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Tetraacetic Acid ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border

Brush-border membrane vesicles from hamster intestine were employed to investigate uptake (binding) of vitamin B12 (B12). Ileal vesicles took up 25 times more B12 than did jejunal vesicles. Uptake of B12 by ileal vesicles was dependent on intrinsic factor (IF) and required Ca2+. Increasing the Ca2+ concentration caused an increase in uptake of B12 reaching a maximum at approximately 8 mM Ca2+. At high Ca2+ concentrations, 6–8 mM, Mg2+ had little effect on uptake of B12. At low Ca2+ concentrations, up to 2 mM, Mg2+ stimulated B12 uptake. Mg2+, Mn2+, and, to a lesser extent, Sr2+ stimulated Ca2+-dependent B12 uptake, but Zn2+, Ba2+, Na+, K+, and La3+ did not. B12 was apparently not metabolized and was bound as IF-B12 complex, which could be removed with (ethylenedinitrilo)tetraacetic acid (EDTA). Our results suggest that two types of divalent cation reactive sites are involved in binding of IF-B12. One is Ca2+ specific. The other is less specific reacting with Mg2+, Mn2+, Sr2+, and perhaps Ca2+ itself, thereby stimulating Ca2+-dependent binding of IF-B12 to its ileal receptor.

Labeling of a glucose binding protein in the rabbit intestinal brush border membrane

1978 ◽  
Vol 56 (5) ◽  
pp. 760-770 ◽  
Author(s):  
J. Lemaire ◽  
D. Maestracci
Keyword(s):  
Molecular Weight ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Electrophoretic Pattern ◽  
Protein Band ◽  
Relative Mobility ◽  
Intestinal Brush Border ◽  
Glucose Binding ◽  
Glucose Binding Protein

Using a double labeling method based on the method of Thomas (Thomas, L. 1973. Isolation of N-ethylmaleimide-labeled phloridzin-sensitive D-glucose binding protein of brash border membrane from rat kidney cortex. Biochim. Biophys. Acta, 291, 454–464.), with radioactive N-ethylmaleimide ([3H]NEM and [14C]NEM) in the presence and absence of D-glucose, a protein band which is periodic acid – Schiff staining insensitive and which has a relative mobility (Rm) of 0.55 (corresponding to a molecular weight of 51 000 daltons) as determined by sodium dodecyl sulfate (SDS) electrophoresis was labeled preferentially.When radioactive p-hydroxymercuriphenylsulfonate ([203Hg]PCMBS) is used in the presence and absence of D-glucose, as described by Smith et al. (SMITH, M. W., FERGUSON, D. R., and BURTON, K. A. 1975. Glucose- and phloridzin-protected thiol groups in pig intestinal brush border membranes. Biochern. J. 147, 617–619.), a protein band which has a relative mobility of 0.62 and a corresponding molecular weight of 42 000 daltons was labeled.Control experiments have shown that increasing concentrations of nonradioactive NEM (0.1–5.0 mM) do not substantially modify the electrophoretic pattern of SDS-solubilized brush border membrane. Nonradioactive PCMBS (0.1–10 mM), on the other hand, modifies the electrophoretic pattern and especially causes a change in relative mobility of the 0.55 protein band which migrates after 1 mM PCMBS treatment with a Rm of 0.62.The effect of 1 mM PCMBS can be reversed by adding L-cysteine or dithiotreitol.Actin extracted from rabbit muscle migrates with the same Rm as the 0.55 protein band in our electrophoretic conditions.

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