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.

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.

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.

Protein digestion in human and rat small intestine: role of new neutral endopeptidases

1988 ◽  
Vol 255 (2) ◽  
pp. G212-G220 ◽  
Author(s):  
D. Guan ◽  
M. Yoshioka ◽  
R. H. Erickson ◽  
W. Heizer ◽  
Y. S. Kim
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biochemical Properties ◽  
Intact Protein ◽  
Human Intestine ◽  
Protein Digestion ◽  
Small Molecular Weight

Two new phosphoramidon-insensitive neutral endopeptidases were identified and partially characterized in the brush-border membrane of rat and human intestine using N-CBZ-L-Ala-L-Arg-L-Arg-4-methoxy-beta-naphthylamide (Z-Ala-Arg-Arg-MNA) and azocasein or alpha-casein as substrates. Activities in the brush-border membrane of both rat and human intestine were maximum at neutral to alkaline pH, were inhibited by metal chelating and thiol reagents, and were insensitive to phosphoramidon. The results also indicate that these endopeptidases are distinct from pancreatic proteases. The biochemical properties of the enzyme hydrolyzing Z-Ala-Arg-Arg-MNA were shown to be different from that hydrolyzing azocasein or alpha-casein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration revealed that several native intact protein substrates were rapidly degraded to small molecular weight peptides and amino acids when incubated with rat or human brush-border membrane preparations. During in vivo intestinal perfusion in rats, 11% of the total administered alpha-casein was hydrolyzed and absorbed by the intestine. The results suggest that phosphoramidon-insensitive endopeptidases in the intestinal brush-border membrane may be of nutritional and physiological importance in protein digestion.

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.

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

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