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 Purification of vacuoles from Neurospora crassa

1981 ◽  
Vol 1 (9) ◽  
pp. 797-806
Author(s):  
L E Vaughn ◽  
R H Davis
Keyword(s):  
Neurospora Crassa ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Velocity Sedimentation ◽  
Equilibrium Density ◽  
Differential Centrifugation ◽  
Sucrose Density Gradient Centrifugation ◽  
Peak Density ◽  
Isopycnic Centrifugation

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.

The Organelle Pathology and Demonstration of Mitochondrial Superoxide Dismutase Deficiency in Two Patients with Dubin–Johnson–Sprinz Syndrome

1978 ◽  
Vol 54 (5) ◽  
pp. 549-553
Author(s):  
T. J. Peters ◽  
Carol A. Seymour
Keyword(s):  
Superoxide Dismutase ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Mitochondrial Superoxide ◽  
Liver Biopsies ◽  
Mitochondrial Superoxide Dismutase

1. Liver biopsies from two patients with the Dubin—Johnson—Sprinz syndrome were subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation and enzymic micro-assays. 2. A selective deficiency of mitochondrial superoxide dismutase has been demonstrated in these patients. 3. The significance of this enzyme deficiency is discussed in relation to the organelle pathology of the syndrome.

scholarly journals Purification of vacuoles from Neurospora crassa.

1981 ◽  
Vol 1 (9) ◽  
pp. 797-806 ◽  
Author(s):  
L E Vaughn ◽  
R H Davis
Keyword(s):  
Neurospora Crassa ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Velocity Sedimentation ◽  
Equilibrium Density ◽  
Differential Centrifugation ◽  
Sucrose Density Gradient Centrifugation ◽  
Peak Density ◽  
Isopycnic Centrifugation

The Neurospora crassa vacuole, defined by its content of basic amino acids, polyphosphate, protease, phosphatases, and alpha-mannosidase, was purified to near homogeneity. The procedure depends upon homogenization of snail gut enzyme-digested cells in a buffer osmotically stabilized with 1 M sorbitol, differential centrifugation of the extract, and sucrose density gradient centrifugation of the organellar pellet. Isopycnic centrifugation of vacuoles in 2.25 M sorbitol-Metrizamide density gradients yielded a peak (density, 1.31 g/cm3) of vacuolar markers coincident with 32P-phospholipids, trichloroacetate-insoluble 14C, and trichloroacetate-soluble 14C. A trail of macromolecular markers in the lighter portions of the gradient reflected, at least in part, heterogeneity of the vacuoles. Almost no contamination by mitochondria or glyoxysomes was detected. Vacuoles were very heterogeneous in size as estimated by velocity sedimentation, but most were larger than mitochondria. Variations of the osmotic strength of the medium were found to alter the equilibrium density of vacuole preparations from 1.06 g/cm3 to over 1.3 g/cm3. This explains the great variation in density reported previously for the "vacuole," the "vesicle," and the "protease particle" of N. crassa, all of which appear to be the same entity.

The subcellular localization of transglutaminase in normal liver and in glucagon-treated and partial hepatectomized rats

1983 ◽  
Vol 3 (12) ◽  
pp. 1085-1090 ◽  
Author(s):  
Sally E. Bruce ◽  
Timothy J. Peters
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Significant Difference

Rat liver was homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density-gradient centrifugation. Transglutaminase, when assayed with putrescine and dimethylcasein as substrates, showed three distinct localizations, cytosol (73%), plasma membrane (20%), and nuclei (7%). The distribution was unaffected by homogenization in the presence of potassium chloride, indicating that the particulate activity was not due to adsorbed cytosolic enzyme. The specific activity and subcellular distribution of transglutaminase in rats which had received intra-peritoneal glucagon, stimulating endocytosis; or which had been subjected to sub-total hepatectomy 2, 16, or 32 h previously, showed no significant difference from control animals.

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