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.

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 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.

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.

Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na(+)-glucose cotransporter

1993 ◽  
Vol 264 (2) ◽  
pp. G294-G299 ◽  
Author(s):  
B. E. Peerce ◽  
M. Cedilote ◽  
R. D. Clarke
Keyword(s):  
Carboxylic Acid ◽  
Glucose Uptake ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Sodium Dodecyl ◽  
Intestinal Brush Border Membrane ◽  
Phlorizin Binding ◽  
Intestinal Brush Border ◽  
Small Intestinal

The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.

scholarly journals Interaction of Escherichia coli K88 antigen with porcine intestinal brush border membranes

1980 ◽  
Vol 29 (3) ◽  
pp. 897-901
Author(s):  
M J Anderson ◽  
J S Whitehead ◽  
Y S Kim
Keyword(s):  
Escherichia Coli ◽  
Brush Border ◽  
Binding Assay ◽  
Polyacrylamide Gel Electrophoresis ◽  
E Coli ◽  
Brush Border Membranes ◽  
Intestinal Epithelia ◽  
Intestinal Brush Border ◽  
Differential Filtration ◽  
Formation Of Complexes

The fimbria-associated Escherichia coli antigen, K88, was purified to homogeneity as determined by polyacrylamide gel electrophoresis and immunodiffusion. This polymeric antigen consists of noncovalently linked subunits, containing little or no carbohydrate, and has a monomeric molecular weight of 23,000. When a binding assay employing differential filtration was used, K88 formed complexes with isolated porcine intestinal brush border membranes. The formation of complexes was inhibited by glycoproteins with terminal N-acetylglucosamine and N-acetylgalactosamine residues and to a lesser extent by free N-acetylhexosamines. These amino sugars may play a role in the interaction of this pathogenic strain of E. coli with the intestinal epithelia of pigs.

scholarly journals The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border

1974 ◽  
Vol 137 (3) ◽  
pp. 489-495 ◽  
Author(s):  
M. A. Kerr ◽  
A. J. Kenny
Keyword(s):  
Molecular Weight ◽  
Brush Border ◽  
Sodium Dodecyl Sulphate ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Atomic Absorption Spectrophotometry ◽  
Sedimentation Equilibrium ◽  
Rabbit Kidney

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [125I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn2+ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn2+- activated endopeptidase to be characterized.

Proteins and enzymes of the brush-border membrane of mouse intestine: influence of organ culture on gel electrophoretic patterns

1982 ◽  
Vol 60 (4) ◽  
pp. 434-443 ◽  
Author(s):  
A. Berteloot ◽  
J. S. Hugon
Keyword(s):  
Membrane Proteins ◽  
Organ Culture ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Specific Activity ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Marker

Purifications of mouse intestinal brush-border membranes from control explants and scrapings of intestinal mucosa have been compared. Based on the specific activity of sucrase used as a specific marker of these membranes, higher purification factors were obtained with control explants (24.7 ± 0.9) as compared with scrapings of intestinal mucosa (14.8 ± 0.9). However, similar patterns of proteins and enzymes were obtained by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis after membrane solubilization by 2% SDS at room temperature. After 24 h of culture, higher molecular weight species of maltase–glucoamylase–isomaltase (band 4), alkaline phosphatase (bands 9–10), and trehalase (band 17) have been observed. Enzyme species appearing in the particulate fraction of culture media were, however, identical with those found at the brush-border membrane level in control explants, except for trehalase. These results are interpreted by considering the possible adsorption of serum components to brush-border membrane proteins. It thus appears that the membrane proteins and enzymes released in the media during organ culture are identical with those synthesized in the tissue in vitro or in vivo.

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