Ectogalactosyltransferase. Presence of Enzyme and Acceptors on the Rat Lymphocyte Cell Surface

Verbert, André; Cacan, René; Montreuil, Jean

doi:10.1111/j.1432-1033.1976.tb10954.x

Cited by 45 publications

(8 citation statements)

References 20 publications

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“…Subcellular fractionation studies from this laboratory favor their specific association with the Golgi apparatus of rat liver cells (28,40), compared with probing spleen lymphocytes (45) and other ceils (46) for ectoenzyme terminal glycosyltransferases that assign some activity to the cell periphery. In our density equilibration experiments, galactosyltransferase and Nacetylglucosaminyltransferase still partly overlap the plasma membrane-associated enzymes in the gradient after addition of digitonin (Figs.…”

Section: Equilibrium Densitymentioning

confidence: 99%

Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages

Darte¹,

Beaufay²

1983

The Journal of Experimental Medicine

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Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid α-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5'-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, α-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum.

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Section: Equilibrium Densitymentioning

confidence: 99%

Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages

Darte¹,

Beaufay²

1983

The Journal of Experimental Medicine

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“…In summary, evidence for a cell surface location of glycosyltransferases is still based on indirect biochemical and autoradiographic data. Glycosyltransferase activities were found associated with plasma membranes upon fractionation (6,12,38), and orientation to the external face was assumed by their ability to glycosylate nonpermeable substrates such as glycoprotein acceptors and sugar-derivatized agarose beads (16,36,37). Autoradiographic evidence that consisted of the electron microscopic demonstration of radioactive substrates incorporated into the plasma membrane also suggested the presence of these enzymes on the cell surface but did not formally prove it (20).…”

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confidence: 99%

Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.

Roth¹,

Lentze²,

Berger³

1985

The Journal of Cell Biology

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Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membraneassociated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.In 1970, Roseman (24) put forward a hypothesis on a possible role of cell surface glycosyltransferases in cell adhesion and recognition. Two reviews on this subject have been published ( 18, 32) and give a comprehensive account of all experimental evidence supporting the original hypothesis. In summary, evidence for a cell surface location of glycosyltransferases is still based on indirect biochemical and autoradiographic data. Glycosyltransferase activities were found associated with plasma membranes upon fractionation (6,12,38), and orientation to the external face was assumed by their ability to glycosylate nonpermeable substrates such as glycoprotein acceptors and sugar-derivatized agarose beads (16,36,37). Autoradiographic evidence that consisted of the electron microscopic demonstration of radioactive substrates incorporated into the plasma membrane also suggested the presence of these enzymes on the cell surface but did not formally prove it (20). The conclusions based on the evidence summarized in the above cited reviews were challenged by Keenan and Morr6 (13) and Deppert et al. (8). At the present time, location ofglycosyltransferase at the plasma membrane is still an open question since no clear-cut in situ demonstration has been provided. Evidence for a putative role in adhesion and recognition, for which a conclusive demonstration of glycosyltransferases on the outer side of the plasma membrane is a prerequisite, remains circumstantial (18).Recently, monospeciflc antibodies against human galactosyltransferase (E.C. 2.4.1.22, 2.4.1.38, 2.4.1.90) became available and were used to localize this enzyme by immu...

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“…UDP-[ 4C]Gal used in the incubation was brought to a specific radioactivity of 200mCi/mmol by the addition of unlabelled UDP-Gal. The incubation medium contained 5 pM-UDP-[14C]Gal, 5mM-MnCl2 and 1 mM-UMP to protect the precursor from the degradation (Verbert et al, 1976). When p-nitrophenyl-p-Dglucosaminide was used as exogenous acceptor, the concentration was 0.5 mg/ml.…”

Section: Preparation Of Nh4ci-treated Thymocytesmentioning

confidence: 99%

Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes

Cacan¹,

Cecchelli²,

Hoflack³

et al. 1984

Biochemical Journal

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Treatment with NH4Cl of mouse thymocytes renders their plasma membrane permeable to sugar nucleotides both inwards and outwards. Using this model, we studied the entry and utilization of CMP-NeuAc, GDP-Fuc and UDP-Gal into intracellular vesicles in situ. It is shown that CMP-NeuAc and GDP-Fuc enter the vesicles in a manner indicating a carrier-mediated transport (substrate saturation curve, inhibition by substrate analogues, temperature dependence) and are entrapped in their uncleaved form. This leads to the formation of an intralumenal pool of these precursors which can be further utilized by the sialyltransferases and fucosyltransferases. The occurrence of an endogenous pool of CMP-NeuAc and GDP-Fuc is demonstrated by the fact that, when the vesicles are disrupted by detergent, the release of the endogenous sugar nucleotides causes an isotopic dilution of the labelled precursors added to measure the glycosyltransferase activities. In contrast, no accumulation of UDP-Gal has been detected, suggesting that transport and transfer reaction are simultaneous events. However, experiments with UDP 2',3'-dialdehyde indicate that UDP-Gal is not transported through the membrane by galactosyltransferase action but by a distinct carrier molecule.

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Ectogalactosyltransferase. Presence of Enzyme and Acceptors on the Rat Lymphocyte Cell Surface

Cited by 45 publications

References 20 publications

Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages

Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages

Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.

Intralumenal pool and transport of CMP-N-acetylneuraminic acid, GDP-fucose and UDP-galactose. Study with plasma-membrane-permeabilized mouse thymocytes

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