Sequential Enzyme and Subcellular Fractionation Studies on Jejunal Biopsy Specimens from Patients with Post-Infective Tropical Malabsorption

1979 ◽  
Vol 56 (5) ◽  
pp. 479-486 ◽  
Author(s):  
T. J. Peters ◽  
P. E. Jones ◽  
G. Wells ◽  
G. G. Cook
Keyword(s):  
Brush Border ◽  
Giardia Lamblia ◽  
Gradient Centrifugation ◽  
Selective Reduction ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Jejunal Biopsy ◽  
Biopsy Specimens ◽  
Glutamyl Transferase

1. Jejunal biopsy specimens were obtained from seven patients with post-infective tropical malabsorption, four of whom were infected with Giardia lamblia. 2. Enzymic analysis of the biopsy homogenates revealed a selective reduction of certain enzymes (alkaline phosphatase, γ-glutamyl transferase) localized at least in part to the brush border. Other enzymes (leucyl-2-naphthylamidase, α-glucosidase) were unaffected. 3. Density gradient centrifugation confirmed the selective decrease in the brush-border activity but the equilibrium density of the organelle was normal. Homogenization studies revealed that the brush border was unusually fragile. 4. Apart from reduced activities of the basal—lateral marker enzyme 5′-nucleotidase and enhanced lysosomal fragility, other organelles showed normal properties. There were no significant differences in any of these parameters in the patients with or without giardiasis. 5. The patients were restudied after their jejunal morphology and clinical features had returned to normal. All these parameters, apart from a persistently reduced activity of 5′-nucleotidase, returned to normal. 6. It is suggested that the principal lesion in patients with post-infective tropical malabsorption affects the cell membrane, specifically affecting the absorptive—digestive capacity of the enterocyte. These findings suggest that there is direct brush-border damage rather than mucosal invasion by bacteria, viruses or Giardia lamblia.

Subcellular Fractionation Studies of the Intestinal Mucosa in Congenital Sucrase—Isomaltase Deficiency

1979 ◽  
Vol 57 (2) ◽  
pp. 181-185 ◽  
Author(s):  
B. T. Cooper ◽  
D. C. A. Candy ◽  
J. T. Harries ◽  
T. J. Peters
Keyword(s):  
Electron Microscopy ◽  
Intestinal Mucosa ◽  
Brush Border ◽  
Enzyme Activities ◽  
Subcellular Fractionation ◽  
The Other ◽  
Marker Enzyme ◽  
Jejunal Biopsy ◽  
Biopsy Specimens ◽  
Highly Sensitive

1. Jejunal biopsy specimens from three children with congenital sucrase—isomaltase deficiency were assayed for disaccharidase activity and were subjected to analytical subcellular fractionation with enzymic microanalysis. 2. By use of the highly sensitive fluorigenic modification of the disaccharidase assay, brush-border sucrase and isomaltase activities were depressed but nevertheless detectable in each child. 3. Apart from the expected decrease in brush-border α-glucosidase activity, the other enterocyte marker-enzyme activities were normal. 4. There were no abnormalities in the enterocytes of any child on analytical subcellular fractionation or on electron microscopy.

Analytical Subcellular Fractionation of Jejunal Biopsy Specimens: Enzyme Activities, Organelle Pathology and Response to Gluten Withdrawal in Patients with Coeliac Disease

1978 ◽  
Vol 55 (3) ◽  
pp. 285-292 ◽  
Author(s):  
T. J. Peters ◽  
P. E. Jones ◽  
G. Wells
Keyword(s):  
Endoplasmic Reticulum ◽  
Coeliac Disease ◽  
Brush Border ◽  
Enzyme Activities ◽  
Subcellular Fractionation ◽  
Density Gradients ◽  
Marker Enzymes ◽  
Jejunal Biopsy ◽  
Essentially Normal ◽  
Biopsy Specimens

1. Portions of jejunal biopsy specimens from patients with coeliac disease were homogenized in isotonic sucrose solution and subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. 2. The activities and distribution of marker enzymes for the principal subcellular organelles were determined by highly sensitive enzymic micro-assays employing fluorigenic and radiolabelled substrates. 3. In untreated coeliac disease the brush border showed decreased activities of five marker enzymes, which were reduced to up to one-eighth of control values. On sucrose density gradients the distinct brush-border peak at density 1.21 was replaced by a broad peak at density 1.16. 4. Two out of three lysosomal enzymes showed increased activities and there was evidence of enhanced fragility of both lysosomes and peroxisomes. 5. There were reduced activities of both mitochondrial and cytosol malate dehydrogenase, with increased activities of cytosol lactate dehydrogenase. 6. The endoplasmic reticulum and basal-lateral membranes were relatively unaffected except that the equilibrium densities were slightly increased and decreased respectively. 7. The patients were treated by gluten withdrawal and subsequent biopsies examined as above. Biopsies from patients treated for 2–4 weeks and who had not yet achieved a morphological improvement showed a partial return to normal of the pathological organelles. 8. Treated patients with partial villus atrophy showed progressively improved enzyme activities and a partial reappearance of the brush-border peak on the sucrose density gradients. 9. Patients whose intestinal mucosa had returned to morphological normality after prolonged gluten withdrawal showed a persistent defect in the brush-border enzyme activities. This was particularly striking for β-glucosidase, which was persistently deficient from the brush-border although approximately one-third of the total activity of this enzyme is normally found in this organelle. 10. The properties and enzyme activities of lysosomes, peroxisomes, endoplasmic reticulum, mitochondria and cytosol were essentially normal in the biopsies from fully treated patients.

Gilbert's Syndrome: Analytical Subcellular Fractionation of Liver Biopsy Specimens. Enzyme Activities, Organelle Pathology and Evidence for Subpopulations of the Syndrome

1979 ◽  
Vol 57 (6) ◽  
pp. 491-497 ◽  
Author(s):  
J. Dawson ◽  
C. A. Seymour ◽  
T. J. Peters
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Liver Biopsy ◽  
Enzyme Activities ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Gilbert’S Syndrome ◽  
Gilbert's Syndrome ◽  
Glutamyl Transferase

1. The hepatic organelle pathology of 12 patients with Gilbert's syndrome was studied by analytical subcellular fractionation in combination with enzymic microanalysis of liver biopsy material. 2. All patients showed increased activities of the endoplasmic reticulum marker enzyme, neutral α-glucosidase. Seven patients showed a decrease in the modal density of the endoplasmic reticulum, from 1·20 to 1·15 g/ml. These patients also showed striking hypertrophy of the hepatocyte smooth endoplasmic reticulum on electron microscopy. The remaining five patients showed normal endoplasmic reticulum density distribution with a peak at 1·20 g/ml, and had normal appearance of the endoplasmic reticulum on electron microscopy. 3. All patients showed increased activity of three lysosomal marker enzymes: N-acetyl-β-glucosaminidase, acid phosphatase and β-glucuronidase. The distribution of these enzymes in the sucrose gradients showed less enzyme in the high-density region of the gradients, indicating a reduced equilibrium density of the lysosomes. Assays of latent and sedimentable N-acetyl-β-glucosaminidase, a measure of lysosomal integrity, were normal. 4. Marker enzyme activities and density gradient distribution of other organelles, including plasma membrane (5′-nucleotidase), mitochondria (malate dehydrogenase), biliary canaliculi (γ-glutamyl transferase) and cytosol (lactate dehydrogenase) were normal. Increased catalase activities were noted.

Galactosyltransferase activity is not localized to the brush border membrane of human small intestine

1986 ◽  
Vol 6 (2) ◽  
pp. 171-175 ◽  
Author(s):  
Frances Boyle ◽  
Susan Snape ◽  
Paul Duane ◽  
Neil Cook ◽  
Timothy Peters
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Sucrose Density Gradient Centrifugation ◽  
Human Small Intestine ◽  
Immunocytochemical Techniques

A recent report [Roth et al. (1985) J. Cell Biol.100: 118–125], using immunocytochemical techniques, calimed that human duodenal galactosyltransferase is located predominantly on the external aspect of enterocyte brush border membranes. Analytical subcellular fractionation by sucrose density gradient centrifugation of human jejunum biopsy homogenates demonstrated that galactosyltransferase activity is localized to the Golgi fraction (equilibrium density of 1.14 g cm−3) and is not found in significant amounts in the brush border membrane (equilibrium density of 1.22 g cm−3).

scholarly journals Analytical subcellular fractionation of needle-biopsy specimens from human liver

1978 ◽  
Vol 174 (2) ◽  
pp. 435-446 ◽  
Author(s):  
T J Peters ◽  
C A Seymour
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Needle Biopsy ◽  
Human Liver ◽  
Resolving Power ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Acid Hydrolases ◽  
Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

Parathyroid hormone-induced translocation of Na-H antiporters in rat proximal tubules

1989 ◽  
Vol 257 (4) ◽  
pp. C637-C645 ◽  
Author(s):  
C. B. Hensley ◽  
M. E. Bradley ◽  
A. K. Mircheff
Keyword(s):  
Parathyroid Hormone ◽  
Density Gradient ◽  
Brush Border ◽  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Subcellular Fractionation ◽  
Proximal Tubules ◽  
Nylon Mesh ◽  
Sequence Of Events ◽  
Distinct Membrane

Parathyroid hormone (PTH) is believed to inhibit bicarbonate reabsorption by inhibiting Na-H antiport activity in proximal tubular brush-border membranes. The sequence of events triggered by PTH was investigated in a crude preparation of proximal tubules obtained by mechanical disruption and filtration through nylon mesh filters. Tubule samples were subjected to analytical subcellular fractionation after 2-, 5-, and 30-min treatments with 1 IU/ml PTH. These PTH-treatment intervals caused 54, 63, and 68% decreases in the Na-H antiport activity of a population of brush-border membrane vesicles that was resolved from a PTH-unresponsive brush-border population by density-gradient centrifugation. The rapid loss of Na-H antiport activity from the responsive population was accompanied by a transient increase in the Na-H antiport activity of a region of the density gradient, designated density window III, which was shown to contain two distinct membrane populations; these populations were both enriched in acid phosphatase activity, and one of them was also an important locus of galactosyltransferase activity. The increase in the Na-H antiport activity of window III accounted for 52% of the activity lost from the PTH-responsive population after 2 min, and for 43% of the activity lost after 5 min, but it was completely abolished after 25 more minutes in the presence of PTH. These observations suggest that PTH triggers a rapid translocation of Na-H antiporters from the microvillus membrane to a distinct membrane domain, where they are subsequently inactivated.

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.

Analytical Subcellular Fractionation of Guinea-Pig Myocardium

1977 ◽  
Vol 53 (1) ◽  
pp. 63-74
Author(s):  
F. J. Bloomfield ◽  
G. Wells ◽  
E. Welman ◽  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Adenosine Triphosphatase ◽  
Small Volume ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Marker Enzyme ◽  
Subcellular Organelles ◽  
Dual Localization ◽  
Glutamyl Transpeptidase

1. Homogenates of guinea-pig left ventricle were fractionated by differential pelleting and by centrifugation on continuous sucrose density gradients. 2. The principal subcellular organelles of myocardium, characterized by their marker enzyme content, were resolved by density gradient centrifugation in a small-volume zonal rotor. The equilibrium densities (p) of the principal organelles are (with marker enzymes in parentheses): sarcolemma, 1·12 (5′-nucleotidase); lysosomes, 1·16 (N-acetyl-β-glucosaminidase); mitochondria, 1·17 (cytochrome oxidase); peroxisomes, 1·18 (catalase); cytosol (lactate dehydrogenase). 3. The subcellular distribution of various adenosine triphosphatase activities and previously unassigned enzymes was determined. Leucyl-β-naphthylamidase and γ-glutamyl transpeptidase showed both cytosol and sarcolemma components. Ca2+-dependent adenosine triphosphatase showed dual localization to the mitochondria and to the sarcolemma.

scholarly journals Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane

1982 ◽  
Vol 203 (2) ◽  
pp. 371-376 ◽  
Author(s):  
L H Lash ◽  
D P Jones
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Specific Activity ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Kidney Cortex ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Lateral Membrane ◽  
Thiol Oxidase

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.

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