Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis

Summary The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. -glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.This work has been supported by grants from France-Québec (M.D., J.H.) and from the Medical Research Council of Canada (D.M., J.S.H.)     

Amphibian intestinal brush border enzymes during thyroxine-induced metamorphosis

The development of intestinal brush border hydrolytic activities has been studied during thyroxine-induced metamorphosis of Rana catesbeiana. Alkaline phosphatase activity peaks at 3 and 10 days after the beginning of the thyroxine treatment. The cytochemical observations concerning alkaline phosphatase activity are in agreement with the biochemical data. At the ultrastructural level, alkaline phosphatase activity is particularly evident on the microvilli membranes of the enterocytes in the primary epithelium after 3 days and in the secondary epithelium after 10 days. gamma-Glutamyltranspeptidase exhibits an increase of activity between 7 and 10 days. On the other hand, glucoamylase, maltase, trehalase and leucylnapthylamidase activities decrease during thyroxine treatment, these enzymatic activities being lower than that normally observed after natural metamorphosis. The present study indicates that even though thyroxine is able to induce the morphological differentiation of the intestinal epithelium this hormone is unable to complete the enzymatic load of the new mucosa.     

Comparative study of some intestinal brush border membrane enzymes during perinatal development of the mouse

Intestinal brush border membrane (bbm) fractions have been isolated from fetal and neonatal mice. The existence of discordant developmental patterns of intestinal enzymatic activity derived from total homogenate and bbm fraction was confirmed. It originates chiefly from two phenomena: (a) variations in the state of purity of brush border fractions, and (b) loss of brush border membrane enzyme activities in supernatant that increases with age. The phenomenon of solubility for glucoamylase and alkaline phosphatase is already present two days before birth.     

Insulin accelerates the development of intestinal brush border hydrolytic activities of suckling mice

The influence of insulin on the postnatal development of intestinal brush border membrane disaccharidase (sucrase, maltase, trehalase, lactase) and peptidase (leucylnaphthylamidase, γ-glutamyltranspeptidase) activities has been studied in mice. At 8 days of age, the animals received either 5, 10, or 12.5 mU insulin/g body wt/day during 3 days. A premature appearance of sucrase activity was noted, the level of sucrase activity being dependent of the amount of insulin injected. Maltase and lactase activities were both increased while trehalase activity was affected only by the highest dose of insulin. The behavior of the two peptidases was quite different as γ-glutamyltranspeptidase was prematurely increased and leucylnaphthylamidase was unaffected by insulin. The hormonal effect is exerted along the entire small intestine. The time course of the responses of the disaccharidases in relationship to cellular migration along the crypt-villus axis has also been studied. By 24 hr after administration of a single injection of 12.5 mU insulin/g body wt, sucrase activity was already present and an increased maltase and trehalase activities were observed. During the subsequent 72 hr no further increase of enzymatic activity was noted even though the epithelial cells are moving up on the villi at a faster rate than in controls, thus indicating that there is no relationship between the enzymatic responses and the cellular migration. The present data show that a premature increase of the circulating level of insulin influences the development of intestinal mucosa in suckling mouse.     

Changes in the functions of the intestinal brush border membrane during the development of the ruminant habit in lambs

1. Brush border membrane vesicles were prepared from lamb enterocytes. These were used to study the changes in the enzyme contents and the transport capacities which occur during the change from a milk to a roughage diet. 2. Na+-dependent transport of D-glucose was present in all regions of the small intestine of pre-ruminant lambs and absent in ruminants. 3. Na+-dependent transport of L-proline was present in all regions of the small intestine irrespective of the age of the animal. 4. Phosphate transport was seen only in the presence of a transmembrane pH gradient (acid outside). The transport was not stimulated by either Na+ or K+. The transport capacity increases 2-fold as the animal becomes ruminant. 5. The activities of lactase and maltase diminished with age. Alkaline phosphatase and aminopeptidase N activities remain constant. Sucrase activity cannot be detected in lambs of any age.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Detergent-induced proteolysis of rabbit intestinal brush border vesicles

Proteolysis of brush border vesicle proteins is induced by detergent solubilisation. This proteolysis is selective in that some of the proteins are more susceptible than others. The rate of induced proteolysis is decreased by decreasing the temperature, has a minimum of about pH 6 and is effectively prevented by a combination of the protease inhibitors, EDTA, diisopropylfluorophosphate and iodoacetamide.     

Uptake of cholesterol by small intestinal brush border membrane is protein-mediated

Absorption of cholesterol by small intestinal brush border membrane from either mixed micelles or small unilamellar vesicles is protein-mediated. It is a second-order reaction. The kinetic data are consistent with a mechanism involving collision-induced transfer of cholesterol. With micelles as the donor particle, there is net transfer of cholesterol while with small unilamellar vesicles as the donor, cholesterol is evenly distributed between the two lipid pools at equilibrium. The cholesterol absorption by brush border membrane from both mixed micelles and small unilamellar vesicles reveals saturation kinetics. Proteolytic treatment of brush border membrane with papain releases about 25% of the total membrane protein. As a result, the cholesterol uptake by brush border membrane changes from a second-order reaction to a first-order one. The reaction mechanism changes from collision-induced cholesterol uptake to a mechanism involving diffusion of monomeric cholesterol through the aqueous phase. The protein(s) released into the supernatant by papain treatment of brush border membrane exhibit(s) cholesterol exchange activity between two populations of small unilamellar vesicles. The supernate-protein(s) bind(s) the spin-labeled cholesterol analogue 3-doxyl-5 alpha-cholestane.     

Characterization of bile acid binding to rat intestinal brush border membranes

Summary Studies were performed to characterize the binding¹ of bile acids to intestinal brush border membranes. Total¹⁴C-taurodeoxycholate binding was: 1) similar for brush borders prepared from jejunum and ileum, 2) linear with respect to monomer concentration, 3) uninhibited by a structural analog, and 4) not depressed by boiling or trypsin. A linear relationship existed between binding and the number of hydrogen bonds formed by a bile acid and the slope of the line corresponded to F of 300 cal/mol. The binding of bile acids to the 105,000×g supernatant fraction of sonicated brush borders was similar to the binding of phospholipid liposomes using gel chromatography. These data suggest that: 1) the kinetics and characteristics of binding of bile acid to ileal brush borders do not reflect the kinetics and characteristics of active ileal transport previously obtained in whole tissue preparations, but instead reflect the kinetics and characteristics of passive jejunal transport; 2) a determinant of binding is hydrogen bonding with water; 3) isolated intact brush borders are relatively polar membranes; and 4) binding to solubilized brush borders may represent partitioning between the aqueous phase and membrane lipid.Part of this work was presented at the National Meeting of the American Federation for Clinical Research, May 2, 1976, Atlantic City, New Jersey.     

Regulation in vitro of brush border myosin by light chain phosphorylation

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.     

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.     

Glycine transport by intestinal brush border vesicles of the amphibian Discoglossus pictus.

1. In order to determine the different components of glycine uptake by the intestine of the frog, Discoglossus pictus, we have used brush border membrane vesicles isolated by a classical precipitation technique. 2. Enzymatic tests showed that a good purification was obtained. The concentration ratio of alkaline phosphatase was 14.8. 3. Glycine entry in vesicles as a function of time, in presence or absence of sodium, indicated an overshoot which decreased when incubation time was prolonged. The overshoot was dependent on the presence of sodium. 4. The nature of the anion associated to sodium had little effect on glycine uptake. Nevertheless, chloride and thiocyanate appeared more efficient than glutarate. 5. The effect of transmembrane potential was studied by using valinomycin associated with a potassium gradient. The addition of this substance stimulated glycine transport by 43%. 6. The transport at different glycine concentrations showed two components: one non-saturable with weak affinity and the other saturable with strong affinity (Kt = 0.338 mM). 7. In conclusion, glycine transport by the brush border of D. pictus intestine presents a saturable component depending on sodium and on transmembrane electrical potential.     

Rap2A links intestinal cell polarity to brush border formation

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P(2) at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.     

Influence of triiodothyronine on the polypeptide composition of the intestinal brush border membrane during amphibian metamorphosis

Brush border fragments (BBF) were isolated from homogenates of intestinal epithelium prepared from four groups of tadpoles: premetamorphic larvae, thyrostatic larvae, spontaneously metamorphosed larvae, and triiodothyronine (T3)-induced froglets. Isolation was accomplished by a combination of both Ca2+ precipitation and differential centrifugation methods. These preparations were routinely enriched seven- to-eleven-fold for the two amphibian brush border marker enzymes, gamma-glutamyltransferase and maltase. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining revealed the presence of a polypeptide of Mr 27,000 only after spontaneous and T3-induced metamorphosis. One-dimensional SDS-PAGE together with lectin staining showed six strongly concanavalin A reactive polypeptides (Mr 52,000, 57,000, 65,000, 80,000, 130,000 and 150,000) in both preparations examined. Immunoblot analyses allowed us to detect in both preparations the presence of villin (Mr 105,000), a cytoskeletal component of microvilli. Two-dimensional isoelectric focusing IEF/SDS-PAGE together with silver staining showed the polypeptides of Mr 41,500, 43,000, 60,500 and 101,000 to be specific components of the primary intestinal epithelium brush border. In contrast six polypeptides of Mr 27,000, 52,000, 58,000, 59,000 and 95,000 were only detected in intestinal BBF after spontaneous and T3-induced metamorphosis. Their presence is under the control of the thyroid hormone. The results provide new insight regarding the subcellular localization of polypeptides whose synthesis changes during spontaneous (Figiel et al., 1987) and T3-induced metamorphosis (Figiel et al., 1989).