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.     

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.)     

Brush border enzymes as markers for ascending pyelonephritis: active immunization with pili

Activities of renal brush border membrane (BBM) enzymes, alkaline phosphatase, maltase and leucine aminopeptidase, were determined in control, pyelonephritic and immunized-infected rats. The activities of all enzymes decreased significantly (P < 0.05) in obstructed kidney while activities of alkaline phosphatase and leucine aminopeptidase increased significantly (P < 0.05) in unobstructed kidney in early stages of infection. The affinity constant (K_m) of all enzymes remained unaltered in control and experimental groups. Significant differences (P < 0.05) in the activities of BBM enzymes of infected and immunized-infected animals suggested a protective role of active immunization with pili.     

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.     

Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium

The brush border of intestinal epithelial cells consists of an array of tightly packed microvilli. Within each microvillus is a bundle of 20-30 actin filaments. The basal ends of the filament bundles are embedded in and interconected by a filamentous meshwork, the terminal web, which lies directly beneath the microvilli. When calcium and ATP are added to isolated brush borders that have been treated with the detergent, Triton X-100, the microvillar filament bundles rapidly retract into and through the terminal web region. Biochemical studies of brush border contractile proteins suggest that the observed microvillar contraction is actomyosin mediated. We have shown previously that the major protein of the brush border's actin (Tilney, L. G., and M. S. Mooseker. 1971. Proc. Natl. Acad. Sci. U. S. A. 68:2611-2615). The brush border also contains a protein with the same molecular weight as the heavy chain subunit of myosin (200, 000 daltons). In addition, preparations of demembranated brush borders exhibit potassium-EDTA ATPase activity of 0.02 mumol phosphate/mg-min (22 degrees C); this assay is diagnostic for myosin-like ATPase isolated from vertebrate sources. Other proteins of the brush border include a 30,000 dalton protein with properties similar to those of tropomyosin, and a protein with the same molecular weight as the Z band protein, alpha-actinin (95,000 daltons). How these observations bear on the basis for microvillar movements in vivo is discussed within the framework of our recent model for the organization of actin and myosin in the brush border (Mooseker, M. S., and L. G. Tilney. 1975. J. Cell Biol. 67:725-743).     

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.     

Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems

The activities of rat intestinal enzymes, sucrase, lactase, maltase, trehalase, γ-glutamyltransferase, leucylnaphthylamide-hydrolyzing activity, and the transport system for glucose follow diurnal rhythms on ad libitum and restricted feeding regimes. In response to 6 days of restricted feeding, food available between 1400 and 1800 Eastern Standard Time, all rhythms shifted in time and the daily levels of activities were changed. Alkaline phosphatase activity followed a diurnal rhythm only in restricted fed animals.In restricted fed rats several activity patterns were observed, some with short periods of maximum activity, 3 h or less, and some with plateaus of maximum activity, 5–9 h long. In respect to the time of day of the synchronizer, sucrase peaked before feeding, glucose transport peaked during feeding, alkaline phosphatase peaked after feeding, and the other enzymes had higher levels of activity before, during and after feeding. The effect of restricted feeding on the daily activity levels were: a decrease in leucylnaphthylamide-hydrolyzing activity, no change in alkaline phosphatase, and increases in the others.These enzyme and transport systems exhibit a large amount of individual regulation or control as reflected by the lack of a uniform activity pattern and response to the synchronizer, and the variation in direction and magnitude of the adaptations to restricted feeding.     

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents. © 1996 Wiley-Liss, Inc.     

Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems.

The activities of rat intestinal enzymes, sucrase, lactase, maltase, trehalase, gamma-glutamyltransferase, leucylnaphthylamide-hydrolyzing activity, and the transport system for glucose follow diurnal rhythms on ad libitum and restricted feeding regimes. In response to 6 days of restricted feeding, food available between 1400 and 1800 Eastern Standard Time, all rhythms shifted in time and the daily levels of activities were changed. Alkaline phosphatase activity followed a diurnal rhythm only in restricted fed animals. In restricted fed rats several activity patterns were observed, some with short periods of maximum activity, 3 h or less, and some with plateaus of maximum activity, 5-9 h long. In respect to the time of day of the synchronizer, sucrase peaked before feeding, glucose transport peaked during feeding, alkaline phosphatase peaked after feeding, and the other enzymes had higher levels of activity before, during and after feeding. The effect of restricted feeding on the daily activity levels were: a decrease in leucylnaphthylamide-hydrolyzing activity, no change in alkaline phosphatase, and increases in the others. These enzyme and transport systems exhibit a large amount of individual regulation or control as reflected by the lack of a uniform activity pattern and response to the synchronizer, and the variation in direction and magnitude of the adaptations to restricted feeding.     

Developmental changes in intestinal brush border enzymes of rats prenatally exposed to ethanol

The activities of lactase, sucrase, alkaline phosphatase (AP) and y-glutamyl transpeptidase (gamma-GTP) were studied in the intestinal brush border membranes of pups born to rat mothers exposed to ethanol (1 ml of 30% ethanol daily during gestation) at different days of postnatal development. The activities of lactase (at day 4-20) and sucrase (at day 20-30) were considerably reduced in response to prenatal exposure to ethanol, while AP (at day 4-30) and gamma-GTP activities were significantly enhanced (p < 0.05) at day 4, 8, 14 and 20, but there was no significant difference by day 30 of postnatal development. The observed changes in enzyme activities were corroborated by Western blot analysis of lactase, sucrase and AP. Kinetic studies revealed a change in Vmax without affecting apparent Km of enzymes under these conditions. The present findings suggest that in utero ethanol exposure to rats is embryotoxic and affects the postnatal development of various brush border enzymes, which persist long after the ethanol was withdrawn prior to birth.     

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.     

Effect of glucose and insulin on small intestinal brush border enzymes in fasted rats

Fasting reduced small intestinal length. It also decreased mucosal weight, DNA and protein content, and concentrations of enterokinase, maltase, and sucrase in both duodenal and jejunal segments. In contrast, the concentrations of lactase and leucine aminopeptidase were not affected. Concomitantly, serum insulin levels dropped to one-fifth of the control levels while serum glucose concentrations showed a lesser degree of reduction. Glucose supplementation alone raised the serum insulin level, prevented the decrease in DNA content, and showed a protective effect on mucosal protein, mucosal weight, mucosal thickness, and villus height. Glucose also protected the sucrase and maltase concentrations; more significantly for maltase in the jejunal segment. Insulin alone, although it increased the serum insulin level to that found with glucose supplementation alone, had no protective effect on the loss in protein, DNA, and most enzymes except for maltase concentration in the jejunal segment. Addition of insulin to glucose did not modify the glucose effect on the contents of DNA, protein, and concentrations of sucrase and maltase. These results suggest that the glucose effect on the mucosa is not mediated by insulin. In addition, the retention of both maltase and sucrase activities through only glucose supplementation suggests the loss of maltase and sucrase in fasting is due to nutrient rather than specific substrate restriction.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.     

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.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.     

The effect of dexamethasone on the development of rat intestinal brush border enzymes in organ culture

The effect of dexamethasone on the evolution pattern of brush border enzymes was examined in the rat jejunum cultured in vitro at different postnatal stages (4 to 21 days). Enzymic activities were analyzed in purified brush border membranes isolated from noncultured intestine and from explants cultured for 24 and 48 hr. The data obtained from this study indicated that dexamethasone exhibits two types of effects on the cultured intestinal tissue: (1) a nonspecific but protective effect against the drastic drop of all enzyme activities as well as against a loss of villus cells observed in control cultures, and (2) a direct and specific effect on precocious induction of sucrase and on stimulation of maltase activity. The SDS-polyacrylamide gel patterns of brush border membrane proteins showed that in the 6-day-old intestine, appearance of sucrase as well as stimulation of maltase activities elicited by dexamethasone were accompanied by a simultaneous appearance or enhancement of the corresponding protein bands. Furthermore, the radioactivity peaks on gels due to the incorporation of ¹⁴C-valine and of ¹⁴C-fucose indicated that dexamethasone induces the synthesis of new proteins or at least the glycosylation of preexisting proteins which may lead to the formation of active maltase and sucrase molecules.     

Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by −20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and ³H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation. © 1996 Wiley-Liss, Inc.     

Effect of heat-stable and heat-labile enterotoxins of Escherichia coli on intestinal brush border membrane enzymes of mice

The activities of intestinal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, lactase, sucrase, gamma-glutamyl transpeptidase and leucine aminopeptidase were determined in intestinal homogenates and purified BBMs from control, heat-stable and heat-labile enterotoxin treated mice. The activities of all the enzymes except lactase were decreased significantly (p less than 0.01) in homogenates while increased significantly (p less than 0.001) in BBMs of experimental groups as compared to controls. Calmodulin activities were increased significantly (p less than 0.01) as compared to control in heat-stable enterotoxin treated mice but remained unaltered in heat-labile enterotoxin treated mice. DNA contents of intestinal homogenates were decreased in experimental groups demonstrating the decrease in cell number in these groups. The altered BBM enzyme activities could not be attributed to changes in calmodulin activities. The increase in enzyme activities in BBMs may reflect a compensatory phenomenon in the remaining cells.     

Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event.

A polyvalent antiserum which precipitates the native, folded, but not the denatured molecular forms of pig intestinal aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) was used to determine the kinetics of polypeptide folding of the two newly synthesized brush border enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a defective cotranslational high-mannose glycosylation, increased the half-time of polypeptide folding to about 12 min for aminopeptidase N as well as for sucrase-isomaltase. Short-pulse experiments suggested that fructose exerts its effect by slowing the rate of glycosylation, making this partially a posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding.