Immunoelectrophoretic studies on pig intestinal brush border proteins.

Brush borders were prepared from pig intestinal mucosa and the membrane proteins solubilized with either Triton X-100 or papain. Proteins, thus released, were used as antigens to raise antisera in rabbits. The immunoglobulin G fractions were isolated and shown by the double layer immunofluorescence staining technique to react only with the brush border region of the enterocyte. The antibodies obtained were used in immunoelectrophoretic studies on the brush border proteins. Eight hydrolytic activities were identified by the use of histo-chemical staining methods. These were the microsomal aminopeptidase (EC 3.4.11.2), aspartate aminopeptidase (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.X), lactase (EC 3.2.1.23), glucoamylase (EC 3.2.1.3), sucrase (EC 3.2.1.48), isomaltase (EC 3.2.1.10) and alkaline phosphatase (EC 3.1.3.1). In addition, at least four faint immunoprecipitates were formed but none of these were identified.     

Dipeptide transport in isolated intestinal brush border membrane.

Transport of glycyl-L-leucine into isolated brush border membrane vesicles was studied. On the basis of the following observations it was postulated that glycyl-L-leucine was transported intact by a specific dipeptide mechanism. (1) The differing time course and Na-+ stimulation of glycine, L-leucine and glycyl-L-leucine. (2) The failure of glycine and L-leucine to inhibit glycyl-L-leucine transport. (3) Initial presence of dipeptide within the vesicle. (4) Inhibition of glycyl-L-leucine uptake by other dipeptides. (5) The occurrence of accelerated amino acid uptake in the presence of the dipeptide.     

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.     

Age related effects of organochlorine insecticide lindane on intestinal brush border membrane in rats.

The effect of oral administration of lindane (gamma-HCH) has been studied on the intestine in 10-day, 20-day and 100-day old rats. In 10 day-old suckling pups exposed to lindane, there was a significant decrease in the activities of sucrase (29%), lactase (20%) and that of alkaline phosphatase (24%) compared to control. Sialic acid content of the brush borders was significantly decreased (29%) in 10-day old as well as in 20- and 100-day old rats (20 and 25% respectively), while fucose content of the membranes was significantly enhanced in all the age groups upon pesticide treatment. Among the brush border lipids, cholesterol content was significantly increased in all the age groups studied, the maximum increase of 35% being observed in 10-day-old rats. Membrane phospholipids were also increased in 20- and 100-day old animals (22% each) on lindane exposure. The present studies indicated that brush border membranes of suckling rat intestine were more susceptible to pesticide induced changes compared to older animals.     

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

ATP-driven copper transport across the intestinal brush border membrane

The divalent metal ion transporter DMT1 is localized in the brush border membrane (BBM) of the upper small intestine and has been shown to be able to transport Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. Belgrade rats have a glycine-to-arginine (G185R) mutation in DMT1, which affects its function. We investigated copper transport with BBM vesicles of Belgrade rats loaded with calcein, which exhibits fluorescence quenching by various metal ions. Transport of copper was disrupted in unenergized BBM vesicle of b/b Belgrade rats, as had been described for iron transport, while +/b vesicles exhibited normal transport by DMT1. When either b/b or +/b vesicles were loaded with ATP and magnesium, similar high-affinity accumulation of copper was observed in both types of vesicles. Thus, brush border membranes possess an ATP-driven, high-affinity copper transport system which could serve as the primary route for copper uptake by the intestine.     

Phase separation of rat intestinal brush border membrane proteins using Triton X-114

Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4 degrees C. Upon phase separation at 32 degrees C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and Ca2+-Mg2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization.     

Distribution of sulfhydryl groups in intestinal brush border membranes. Localization of side-chains essential for glucose transport and phlorizin binding

1. Brush border membrane vesicles from rabbit small intestine were found to contain 46 nmol SH groups/mg protein, 52% of which could react with 4,4'-dithiodipyridine, a membrane permeating probe. Only 18% of the total SH-groups reacted with the impermeant probe 5,5'-dithiobis(2-nitrobenzoic acid), indicating that only this fraction is externally located. 2. Brush border membrane vesicles could be disrupted by a gentle treatment with deoxycholate, releasing most of their electron-dense core material. In deoxycholate-treated vesicles most of the SH groups that reacted with 4,4'-dithiodipyridine react with 5,5'-dibiobis(2-nitrobenzoic acid), suggesting that both membrane surfaces became exposed to the extravesicular medium. 3. In intact vesicles (1.2 mg protein/ml), the binding of phlorizin (a competitive inhibitor of the monosaccharide transport system) was 50% inhibited by 67 microM of the penetrating organomercurial p-chloromercuribenzoate, but was about ten times less sensitive to the poorly permeating p-chloromercuriphenylsulfonate. In contrast, binding of phlorizin to leaky (deoxycholate-treated) membranes was equally susceptible to either reagent. 4. Mercurial inhibition of phlorizin binding could be reversed by dithioerythritol in both sealed and leaky membranes, whereas the less permeant thiol L-glutathione (reduced form) could only revert the inhibition in leaky membranes.     

Enzymic solubilization of the human intestinal brush border membrane enzymes

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, γ-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush border membrane vesicles by various enzymes (especially pancreatic proteases) have been studied.The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases.Electron microscopy of brush border membrane vesicles demonstrates “knob-like” structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of th enzymic activities with the removal of the particles.The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

Integration of alkaline phosphatase in the intestinal brush border membrane

Alkaline phosphatase has been solubilized from porcine intestinal mucosa by two different methods: treatment of the mucosa by Emulphogen BC 720 and papain hydrolysis of enterocyte brush border membrane vesicles. Two different enzyme forms have been obtained by these methods.The two enzyme forms (‘detergent form’ and ‘papain form’) have been purified to homogeneity by similar techniques and exhibit closely related molecular characteristics. However, the detergent form displays a hydrophobic behaviour and aggregates in media free of detergent. The two forms can be differentiated by their electrophoretic mobility on polyacrylamide gel in the absence of sodium dodecyl sulphate.By electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate, it has been shown that the detergent and papain forms of alkaline phophatase are dimers consisting of two apparently identical subunits whose molecular weights are 64 000 and 61 000, respectively. The difference between these molecular weights has been attributed to the existence of a hydrophobic region in the detergent form which is present on each subunit.     

Myosins and membrane trafficking in intestinal brush border assembly

Myosins are a class of motors that participate in a wide variety of cellular functions including organelle transport, cell adhesion, endocytosis and exocytosis, movement of RNA, and cell motility. Among the emerging roles for myosins is regulation of the assembly, morphology, and function of actin protrusions such as microvilli. The intestine harbors an elaborate apical membrane composed of highly organized microvilli. Microvilli assembly and function are intricately tied to several myosins including Myosin 1a, non-muscle Myosin 2c, Myosin 5b, Myosin 6, and Myosin 7b. Here, we review the research progress made in our understanding of myosin mediated apical assembly.     

Ion permeability of rabbit intestinal brush border membrane vesicles

Summary The ion permeability of rabbit jejunal brush border membrane vesicles was studied by measuring unidirectional fluxes with radioactive tracers and bi-ionic diffusion potentials with the potential-sensitive fluorescent dye, diS–C₃-(5). Tracer measurements provide estimates of the absolute magnitudes of permeability coefficients, while fluorescence measurements provide estimates of relative and absolute ion permeabilities. The magnitudes of the permeability coefficients for Na⁺, K⁺, Rb⁺, and Br^– were approximately 5 nanoliters/(mg protein × sec) or 10^–5 cm/sec as determined by radioactive tracer measurements. The apparent selectivity sequence, relative to Na⁺, as determined by bi-ionic potential measurements was: F^–, isetheionate, gluconate, choline (<0.1)+(1.0)–(1.5)=NO ₃ ^– (1.5)–(2.3)+(2.4)+(2.5)+(2.6)+(3.9) 4 ^– +(12)–(40). The origin of this selectivity sequence and its relationship to the ion permeability of the brush border membrane in the intact epithelium are discussed.