Analytical Subcellular Fractionation Studies on Enterocytes from the Jejunum and Ileum of the Rat and Some Properties of Brush-Border Alkaline Phosphatase

1978 ◽  
Vol 55 (2) ◽  
pp. 157-165
Author(s):  
R. M. Batt ◽  
T. J. Peters
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Subcellular Fractionation ◽  
Proximal Jejunum ◽  
Marker Enzymes ◽  
Brush Border Enzymes ◽  
Mechanical Disruption ◽  
Brush Borders ◽  
Mitochondrial Malate Dehydrogenase ◽  
Isopycnic Centrifugation

1. Enterocytes, isolated from the proximal jejunum and distal ileum of the rat, were homogenized and their organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal organelles, RNA and protein were determined in the sucrose gradients and related to the activities per enterocyte. 2. In the jejunum the modal equilibrium densities of the various organelles were: brush borders (1.20), lysosomes (1.20), peroxisomes (1.19), mitochondria (1.17) and basal-lateral membranes (1.13). These values were not significantly different in the ileum. The activities of brush-border enzymes, soluble and mitochondrial malate dehydrogenase, soluble and membrane-associated lactate dehydrogenase and particulate protein content, however, were greater in the jejunal than the ileal enterocytes. 3. Detergent exposed latent alkaline phosphatase activity in jejunal enterocytes and indicated that this enzyme is present not only in the brush border but also in the basal-lateral membrane and soluble fractions of the cell. 4. Isolated jejunal brush-border preparations showed latent activities of both alkaline phosphatase and γ-glutamyltransferase whereas the activities of α-glucosidase and leucyl-β-naphthylamidase were not affected by detergent. Mechanical disruption of these preparations suggested the presence of two forms of alkaline phosphatase in the brush border and provides a technique to assess membrane fragility.

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.

Different diuresis-dependent excretions of urinary enzymes: N-acetyl-beta-D-glucosaminidase, alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase.

1986 ◽  
Vol 32 (3) ◽  
pp. 529-532 ◽  
Author(s):  
K Jung ◽  
G Schulze ◽  
C Reinholdt
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Excretion Rate ◽  
Urine Flow ◽  
High Intake ◽  
Brush Border Enzymes ◽  
Urinary Creatinine ◽  
Gamma Glutamyltransferase ◽  
Alanine Aminopeptidase ◽  
Brush Border Enzyme

Abstract We studied how much of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) and of the brush-border enzymes alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and gamma-glutamyltransferase (EC 2.3.2.2) was excreted in urine over 8 h after a high intake of fluid (22 mL per kilogram of body weight). The hourly excretion of all four enzymes increased with the increasing urine flow rate. The excretion rate of the brush-border enzymes was more markedly influenced than that of N-acetyl-beta-D-glucosaminidase. By relating the enzyme excretion to urinary creatinine we could reduce the variability of brush-border enzyme output and could completely compensate for the effect of diuresis on the excretion of N-acetyl-beta-D-glucosaminidase.

Analytical Subcellular Fractionation Studies on Rat Liver and on Isolated Jejunal Enterocytes with Special Reference to the Separation of Lysosomes, Peroxisomes and Mitochondria

1976 ◽  
Vol 50 (5) ◽  
pp. 355-366 ◽  
Author(s):  
T. J. Peters ◽  
H. Shio
Keyword(s):  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Low Molecular Weight ◽  
Rat Jejunum ◽  
Marker Enzymes ◽  
Good Separation ◽  
Subcellular Organelles ◽  
Rat Liver Cells ◽  
Isopycnic Centrifugation

1. Enterocytes were isolated from rat jejunum and characterized morphologically. 2. Attempts to separate the enterocyte subcellular organelles, characterized by their marker enzymes, with isopycnic centrifugation were unsuccessful but good separation of peroxisomes, lysosomes and mitochondria was achieved by sedimentation through a shallow sucrose density gradient with a superimposed inverse gradient of low-molecular-weight dextran. 3. The properties and enzyme activities of the principal subcellular organelles in rat liver cells and enterocytes were compared.

scholarly journals ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF BRUSH BORDERS FROM RABBIT KIDNEY

1970 ◽  
Vol 47 (3) ◽  
pp. 637-645 ◽  
Author(s):  
Sosamma J. Berger ◽  
Bertram Sacktor
Keyword(s):  
Brush Border ◽  
Renal Cortex ◽  
Biochemical Characterization ◽  
Rabbit Kidney ◽  
Renal Tubules ◽  
Marker Enzymes ◽  
Alkaline Phosphatases ◽  
Brush Borders ◽  
Specific Activities

A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated. The procedure was monitored by phase and electron microscopy and marker enzymes, i.e. ATP:NMN adenylyl transferase, nuclear; cytochrome oxidase, mitochondrial; ß-glucuronidase, lysosomal; and glucose-6-Pase, microsomal; and indicated an essentially pure preparation of brush borders. The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders. Maltase was also found; the specific activities of the two enzymes in the brush borders were increased 10- to 20-fold. Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent. It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na+ + K+) ATPase, an oligomycin-insensitive Mg++ ATPase, and a Ca++-activated ATPase. Alkaline phosphatases, dephosphorylating ß-glycero-P, and trehalose-6-P were also present. The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations; however, activities were found in other subcellular fractions of the renal cortex. Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions. Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex.

scholarly journals The subcellular localization of di- and tri-peptide hydrolase activity in guinea-pig small intestine

1970 ◽  
Vol 120 (1) ◽  
pp. 195-203 ◽  
Author(s):  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Brush Border ◽  
Soluble Fraction ◽  
Subcellular Fractionation ◽  
Border Region ◽  
Protein Digestion ◽  
Kinetic Assay ◽  
Brush Borders

1. Two different subcellular fractionation techniques were applied to guinea-pig intestinal mucosa and the composition of the brush borders prepared by the two methods were compared. 2. By using a kinetic assay system the subcellular distribution of activity against ten dipeptides and five tripeptides was studied. 3. Only small amounts (5–10%) of activity against dipeptides were found in the brush-border region, the enzymes being concentrated in the cytosol. 4. Significant amounts (10–60%) of activity against tripeptides were found in the brush border with the remainder largely present in the soluble fraction. 5. The relevance of these studies to the localization in vivo and the possible role of peptidases in protein digestion is discussed.

scholarly journals Characterization of brush borders purified in iso-osmotic medium and microvillar membranes subfractionated from mouse small intestine

1981 ◽  
Vol 196 (3) ◽  
pp. 669-673 ◽  
Author(s):  
M Fujita ◽  
H Ohta ◽  
T Uezato
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gamma Glutamyl Transpeptidase ◽  
Electrophoretic Patterns ◽  
Mouse Small Intestine ◽  
Brush Borders ◽  
Typical Morphology ◽  
Glutamyl Transpeptidase

Brush borders free of nuclei were isolated by repeated homogenization and centrifugation in iso-osmotic medium. They showed typical morphology under electron microscopy. The mean recovery and enrichment of alkaline phosphatase activity in the brush-border fraction were 50% and 17.5-fold respectively. gamma-Glutamyl transpeptidase showed a close parallelism with alkaline phosphatase and sucrase in subcellular distribution. Microvillar membranes were purified from isolated brush borders; they showed a further enrichment for alkaline phosphatase and were composed of homogeneous vesicles. Both brush-border and microvillar-membrane preparations were analysed for contamination by basolateral and endoplasmic-reticular membranes. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the microvillar-membrane preparation in six different systems revealed approx. 40 components in the mol.wt. range 15 000-232 000. They were grouped into seven major classes on the basis of molecular weight and electrophoretic patterns.

scholarly journals Phosphatidylinositol kinase and diphosphoinositide kinase of rat kidney cortex: properties and subcellular localization

1976 ◽  
Vol 160 (1) ◽  
pp. 97-105 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Brush Border ◽  
Rat Kidney ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Marker Enzymes ◽  
Rat Kidney Cortex ◽  
Phosphatidylinositol Kinase ◽  
Golgi Membranes

The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.

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.

scholarly journals Plasma lipopolysaccharide level and enterocyte brush border enzymes in gnotobiotic piglets infected with Salmonella typhimurium

2012 ◽  
Vol 47 (No. 10 - 11) ◽  
pp. 289-294
Author(s):  
I. Trebichavský ◽  
H. Kozáková ◽  
IŠplíchal
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border ◽  
Virulent Strain ◽  
Systemic Infection ◽  
Gamma Glutamyl Transpeptidase ◽  
Brush Border Enzymes ◽  
Dipeptidylpeptidase Iv ◽  
Glutamyl Transpeptidase ◽  
Rough Mutant ◽  
Gnotobiotic Piglets

Gnotobiotic piglets were orally infected either with the virulent LT2 strain or the non-pathogenic SF1591 rough mutant of Salmonella enterica serotype Typhimurium. They were sacrificed 6 or 24 h after the infection. All piglets infected for 24 h developed systemic infection with an increase of plasma lipopolysaccharide. Infection with the virulent strain caused a significant decrease (P < 0.001) of gamma-glutamyl transpeptidase (GGT) activity in the enterocyte brush border of both the jejunum and ileum, infection with the rough mutant caused a decrease of GGT activity in the ileum only. The activities of other brush border enzymes (lactase, sucrase, glucoamylase, alkaline phosphatase and dipeptidylpeptidase IV) did not change significantly after infection.

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