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.

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.

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.

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.

Brush border enzyme activities in the small intestine after long-term gliadin feeding in animal models of human cœliac disease

1998 ◽  
Vol 43 (5) ◽  
pp. 497-500 ◽  
Author(s):  
H. Kozáková ◽  
R. Štěpánková ◽  
J. Kolínská ◽  
M. A. Farré ◽  
D. P. Funda ◽  
...  
Keyword(s):  
Small Intestine ◽  
Animal Models ◽  
Coeliac Disease ◽  
Brush Border ◽  
Enzyme Activities ◽  
Brush Border Enzyme

scholarly journals Integrated stereological and biochemical studies on hepatocytic membranes. I.V. Heterogeneous distribution of marker enzymes on endoplasmic reticulum membranes in fractions.

1980 ◽  
Vol 85 (3) ◽  
pp. 577-586 ◽  
Author(s):  
R P Bolender ◽  
D Paumgartner ◽  
D Muellener ◽  
G Losa ◽  
E R Weibel
Keyword(s):  
Endoplasmic Reticulum ◽  
Surface Area ◽  
Enzyme Activities ◽  
Membrane Surface ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Heterogeneous Distribution ◽  
Membrane Surface Area ◽  
Total Membrane ◽  
Nadph Cytochrome C Reductase

The purpose of the study was to consider quantitatively the relationships between the surface area of the endoplasmic reticulum (ER) and constituent marker enzyme activities, as they occur in fractions collected from rat liver homogenates. The ER surface area was estimated in five membrane-containing fractions by use of a combined cytochemical-stereological technique (5), while, at the same time, ER marker enzymes were assayed biochemically. Fraction/homogenate recoveries for the ER enzymes averaged 100%, total membrane surface area 98%, and ER surface area 96%. Relative specific activities, which compare the relative amounts of ER marker enzyme activities to the relative ER surface area in the membrane-containing fractions, indicate variable distributions for glucose-6-phosphatase and NADPH cytochrome c reductase, but not for esterase.

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.

Effect of Bile-Duct Ligation on Organelle Marker Enzymes in the Liver and Serum of Rats

1975 ◽  
Vol 48 (4) ◽  
pp. 307-313
Author(s):  
T. J. Peters ◽  
G. Neale ◽  
J. R. Heath
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Bile Duct ◽  
Enzyme Activities ◽  
Liver Tissue ◽  
Bile Duct Ligation ◽  
Monoamine Oxidase A ◽  
Mitochondrial Enzyme ◽  
Marker Enzymes ◽  
Duct Ligation

1. Marker enzymes for the principal subcellular organelles of rat liver were asayed in the liver of rats 1 day and 8 days after bile-duct ligation or after laparotomy as a control procedure. 2. The microsomal enzymes in liver tissue showed complex changes. Benz[α]pyrene hydroxylase activity, predominantly found in the smooth endoplasmic reticulum, was decreased. Glucose 6-phosphatase activity and ribonucleic acid, which are localized predominantly in the rough endoplasmic reticulum, were increased. 3. The plasma membrane enzyme, alkaline phosphatase, increased in activity after bile-duct ligation. 4. No changes in mitochondrial enzyme activities were noted after 1 day but there was a 50% reduction 8 days after ligation. Lysosomal enzyme activities did not change in the liver tissue. 5. Liver catalase and d-amino acid oxidase activities showed a slight increase at 1 day post-ligation but a significant fall by 8 days. 6. Lactate dehydrogenase, a cytosol enzyme, showed a decrease in activity after 1 day but an increase in tissue activities 8 days after ligation. 7. Serum activities of mitochondrial, plasma membrane, microsomal, lysosomal and cytosol marker enzymes tended to increase post-ligation, particularly at 8 days. 8. Monoamine oxidase, a predominantly mitochondrial enzyme, was greatly elevated in the serum after 1 day but had returned to normal activities by 8 days.

Effect of reversible and irreversible ischemia on marker enzymes of BBM from renal cortical PT subpopulations

1997 ◽  
Vol 273 (6) ◽  
pp. F849-F856 ◽  
Author(s):  
Syed Jalal Khundmiri ◽  
Mohammed Asghar ◽  
Farah Khan ◽  
Samina Salim ◽  
Ahad Noor Khan Yusufi
Keyword(s):  
Renal Failure ◽  
Brush Border ◽  
Enzyme Activities ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Supernatant Fraction ◽  
Marker Enzymes ◽  
Rate Of Recovery ◽  
Membrane Bound ◽  
Partial Reversal

The effect of the reversible and relatively irreversible ischemia induced acute renal failure (ARF) in the activities of alkaline phosphatase (AlkPase) and γ-glutamyltransferase (GGTase) after early (15–30 min) and prolonged (45–60 min) ischemia in the homogenates, and the brush-border membranes (BBM) from rat renal whole, superficial (SC), and juxtamedullary (JMC) cortices were studied. The enzyme activities declined progressively in proportion to the duration of ischemia. Early blood reflow of 15 min to the ischemic rats caused a further decrease in the enzyme activities. However, prolonged reflow (up to 120 min) resulted in partial reversal of the ischemic effect in the early but not in the prolonged ischemic rats. The decrease in the enzyme activities was due to the loss of membrane-bound enzyme components from the damaged BBM into the supernatant fraction as membrane-free enzymes. The activities of AlkPase and GGTase were significantly more decreased by the ischemia in the brush-border membrane vesicles (BBMV)-JMC than in BBMV-SC. The rate of recovery due to reflow for AlkPase was greater in BBMV-SC than apparently for GGTase in BBMV-JMC in early ischemic (15–30 min) rats.

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