scholarly journals Lactase-phlorizin hydrolase and aminopeptidase N are differentially regulated in the small intestine of the pig

1993 ◽  
Vol 295 (1) ◽  
pp. 177-182 ◽  
Author(s):  
N Torp ◽  
M Rossi ◽  
J T Troelsen ◽  
J Olsen ◽  
E M Danielsen
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Aminopeptidase N ◽  
Proximal Jejunum ◽  
Brush Border Enzymes ◽  
Sharp Maximum ◽  
Pancreatic Proteases ◽  
Mucosal Explants

The longitudinal expression of two brush-border enzymes, lactase-phlorizin hydrolase (EC 3.2.1.23/62) and aminopeptidase N (EC 3.4.11.2), was studied in the small intestine of the post-weaned pig. Whereas the level of mRNA, encoding aminopeptidase N (relative to that of beta-actin), only varied moderately from the duodenum to the terminal ileum, the amount of lactase-phlorizin hydrolase mRNA exhibited a sharp maximum in the proximal jejunum. For both enzymes, the level of protein synthesis, studied in cultured mucosal explants, correlated well with the level of mRNA, and no major variation in post-translational processing or intracellular transport was observed along the intestine. The mRNA/specific-activity ratio for both enzymes was markedly (3-5-fold) higher in the duodenum and proximal jejunum, compared with the ileum. This indicates an increased proximal turnover rate, most likely caused by the presence in the gut lumen of pancreatic proteases. In neonatal animals, the level of mRNA for lactase-phlorizin hydrolase in both proximal and distal regions of the intestine was of the same magnitude as in the proximal jejunum of the post-weaned pigs. Our results point to two mechanisms that affect the expression of lactase-phlorizin hydrolase in the pig during development: (1) a primary regulation at the level of mRNA (predominantly in the ileum); (2) an increased rate of turnover of the enzyme, mainly in the duodenum and proximal jejunum, and most likely due to an increased secretion into the gut lumen of pancreatic proteases (a mechanism also affecting aminopeptidase N and probably other brush-border enzymes as well).

scholarly journals Galectin-4 and Small Intestinal Brush Border Enzymes Form Clusters

1997 ◽  
Vol 8 (11) ◽  
pp. 2241-2251 ◽  
Author(s):  
E. Michael Danielsen ◽  
Bo van Deurs
Keyword(s):  
Brush Border ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Aminopeptidase N ◽  
Intestinal Lumen ◽  
Immunogold Electron Microscopy ◽  
Brush Border Enzymes ◽  
Intestinal Brush Border ◽  
Mucosal Explants

Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed that galectin-4 is indeed an intestinal brush border protein; we also localized galectin-4 throughout the cell, mainly associated with membraneous structures, including small vesicles, and to the rootlets of microvillar actin filaments. This was confirmed by subcellular fractionation, showing about half the amount of galectin-4 to be in the microvillar fraction, the rest being associated with insoluble intracellular structures. A direct association between the lectin and aminopeptidase N was evidenced by a colocalization along microvilli in double immunogold labeling and by the ability of an antibody to galectin-4 to coimmunoprecipitate aminopeptidase N and sucrase-isomaltase. Furthermore, galectin-4 was released from microvillar, right-side-out vesicles as well as from mucosal explants by a brief wash with 100 mM lactose, confirming its extracellular localization. Galectin-4 is therefore secreted by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly “trapped” by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border enzymes prevents it from being released from the enterocyte into the intestinal lumen.

The Effects of Prednisolone on the Rat Enterocyte at a Subcellular Level

1978 ◽  
Vol 55 (5) ◽  
pp. 435-443
Author(s):  
R. M. Batt ◽  
G. Wells ◽  
T. J. Peters
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Equilibrium Density ◽  
Proximal Jejunum ◽  
Brush Border Enzymes ◽  
Brush Borders ◽  
Brush Border Enzyme ◽  
Mitochondrial Malate Dehydrogenase ◽  
Subcellular Level ◽  
Isopycnic Centrifugation

1. Enterocytes, isolated from the proximal jejunum and distal ileum of normal and prednisolone-treated rats, were homogenized and fractionated by isopycnic centrifugation on sucrose density gradients. The distributions of marker enzymes for the principal subcellular organelles, RNA and protein were determined and related to the activities per enterocyte. 2. In enterocytes from the jejunum and ileum of prednisolone-treated animals the activities of particulate brush-border enzymes and of both soluble and mitochondrial malate dehydrogenase were increased compared with those of the control system. The equilibrium density of the brush borders was enhanced in the prednisolone-treated jejunum. The modal densities of the other organelles were unaltered by prednisolone administration. 3. There was a large increase in the total RNA content of enterocytes from the jejunum and ileum of prednisolone-treated animals. This was predominantly associated with a distinct particulate component, indicative of a proliferation of the rough endoplasmic reticulum and consistent with an enhanced rate of protein synthesis. 4. Studies of latent brush-border enzyme activities, the mechanical fragility of isolated brush borders and electron microscopy suggest that steroid administration results in no marked alterations in the gross conformation of the brush- border membrane or in the orientation of the enzymes within the membrane.

scholarly journals The Influence of Peptidases in Intestinal Brush Border Membranes on the Absorption of Oligopeptides from Whey Protein Hydrolysate: An Ex Vivo Study Using an Ussing Chamber

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1415
Author(s):  
Luísa Ozorio ◽  
Caroline Mellinger-Silva ◽  
Lourdes M. C. Cabral ◽  
Julien Jardin ◽  
Gaelle Boudry ◽  
...  
Keyword(s):  
Whey Protein ◽  
Brush Border ◽  
Ex Vivo ◽  
Protein Hydrolysate ◽  
Ussing Chamber ◽  
Proximal Jejunum ◽  
Peptidase Activity ◽  
Ex Vivo Model ◽  
Brush Border Enzymes ◽  
Whey Protein Hydrolysate

For many years, it was believed that only amino acids, dipeptides, and tripeptides could be absorbed and thus reach the bloodstream. Nowadays, the bioavailability of oligopeptides is also considered possible, leading to new research. This pilot study investigates the activity of brush border enzymes on undigested whey protein hydrolysate (WPH) and on simulated intestinal digested (ID) whey hydrolysate and the subsequent absorption of resultant peptides through the proximal jejunum of a 7-week old piglet setup in an Ussing chamber model. Amongst all samples taken, 884 oligopeptides were identified. The brush border peptidase activity was intense in the first 10 min of the experiment, producing several new peptides in the apical compartment. With respect to the ID substrate, 286 peptides were detected in the basolateral compartment after 120 min of enzyme activity, originating from β-lactoglobulin (60%) and β-casein (20%). Nevertheless, only 0.6 to 3.35% of any specific peptide could pass through the epithelial barrier and thus reach the basolateral compartment. This study demonstrates transepithelial jejunum absorption of whey oligopeptides in an ex vivo model. It also confirmed the proteolytic activity of brush border enzymes on these oligopeptides, giving birth to a myriad of new bioactive peptides available for absorption.

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine

2015 ◽  
pp. 59-118 ◽  
Author(s):  
Diane Hooton ◽  
Roger Lentle ◽  
John Monro ◽  
Martin Wickham ◽  
Robert Simpson
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Brush Border Enzymes

scholarly journals Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases

2020 ◽  
Vol 19 (12) ◽  
pp. 2104-2114
Author(s):  
Matthias Schittmayer ◽  
Nemanja Vujic ◽  
Barbara Darnhofer ◽  
Melanie Korbelius ◽  
Sophie Honeder ◽  
...  
Keyword(s):  
Small Intestine ◽  
Lipid Droplets ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Relative Activity ◽  
Proximal Jejunum ◽  
Serine Hydrolase ◽  
Spatially Resolved ◽  
Molecular Events ◽  
Labeling Approach

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

scholarly journals The effect of starvation on the control of phosphofructokinase activity in the epithelial cells of the rat small intestine

1983 ◽  
Vol 210 (1) ◽  
pp. 129-135 ◽  
Author(s):  
A Jamal ◽  
G L Kellett
Keyword(s):  
Small Intestine ◽  
Epithelial Cells ◽  
Terminal Ileum ◽  
Specific Activity ◽  
Utilization Rate ◽  
Proximal Jejunum ◽  
Rat Small Intestine ◽  
Humoral Factors ◽  
Specific Activities ◽  
Regulatory Properties

1. The effect of depriving rats of food for 48 h on the specific activity of phosphofructokinase in the epithelial cells of the small intestine and on the regulatory properties of the enzyme displayed in crude (particle-free) mucosal extracts was studied. 2. The specific activity of phosphofructokinase, measured under optimal conditions at pH8, in the mucosa of fed rats showed a negative aboral gradient along the intestine, decreasing from 15.2 +/- 1.2 units (mumol/min)/g wet wt. in the proximal jejunum to 4.6 +/- 1.2 units/g wet wt. in the terminal ileum. 3. After starvation, the gradient was diminished, but not abolished; the diminution in gradient was due almost exclusively to a decrease in the specific activity of phosphofructokinase in the proximal jejunum by about 30%, there being no change in the terminal ileum. 4. In fed rats, the susceptibility of phosphofructokinase to inhibition by ATP, when assayed in crude mucosal extracts under suboptimal conditions, was independent of length along the small intestine; the ratio of the activity observed at pH 7.0 in the presence of 0.5 mM-fructose 6-phosphate and 2.5 mM-ATP to the optimal activity at pH 8, v0.5/V, was 0.36 +/- 0.05 in the proximal jejunum and 0.42 +/- 0.07 in the terminal ileum. 5. After starvation, the susceptibility of phosphofructokinase to inhibition by ATP was increased and was again found to be independent of length along the small intestine: after starvation, v0.5/V was 0.19 +/- 0.04 and 0.20 +/- 0.07 for the proximal jejunum and the terminal ileum respectively. 6. Re-feeding of previously starved rats on a high-carbohydrate diet overnight for 16 h restored both the specific activities of phosphofructokinase and its susceptibility to inhibition by ATP to normal values for fed rats. 7. The data support the idea that the specific activities and the regulatory properties of phosphofructokinase in the epithelial cells of rat small intestine are mediated by distinct humoral factors. 8. The changes in glucose utilization rate of the jejunum when rats are starved can in principle be accounted for by a combination of changes in the specific activity and in the regulatory properties of mucosal phosphofructokinase.

Antibodies in the small intestine: mucosal synthesis and deposition of anti-glycosyl IgA, IgM, and IgG in the enterocyte brush border

2006 ◽  
Vol 291 (1) ◽  
pp. G82-G90 ◽  
Author(s):  
Gert H. Hansen ◽  
Lise-Lotte Niels-Christiansen ◽  
Lissi Immerdal ◽  
E. Michael Danielsen
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Fluorescent Protein ◽  
Basolateral Membrane ◽  
Protein G ◽  
Isolated Cells ◽  
Double Labeling ◽  
Protective Function ◽  
Immunoglobulin Receptor ◽  
Mucosal Explants

Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and β2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, β2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that “anti-glycosyl” antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface.

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