Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.99.6 (sialyltransferase)
 1,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our previously described immunoadsorption method for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters from 3T3-L1 adipocytes has been improved in two ways. First, the minimal number of g minutes required to sediment the plasma membranes from the cell homogenate has been determined and, as a result, the supernatant used for immunoadsorption in the new procedure contained twice as much of the intracellular transporters. Second, the immunoadsorption has been performed with affinity-purified antibodies directed against the carboxy terminal peptide of the transporter, rather than against the entire protein. 10(7) cells (10 mg protein) yielded about 12 micrograms of vesicular protein and 11 micrograms of vesicular phospholipid. The transporter constituted 3% of the protein in the vesicles; this amount equates to approx. eight copies of the transporter per 50 nm vesicle. The polypeptide composition of the vesicles was determined by gel electrophoresis and protein staining. Major components, other than the glucose transporter, are polypeptides of Mr 270,000, 245,000, 165,000 and 115,000. The vesicles contained several phosphoproteins; the major ones have a Mr of 245,000, 190,000, 115,000 and 25,000. Insulin treatment of adipocytes did not significantly change the phosphoprotein composition of the vesicles. The vesicles were not enriched in the Golgi marker enzyme, galactosyltransferase. The cellular content of the marker for the trans-Golgi reticulum, sialyltransferase, was too low to detect.
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PMID:Characterization of vesicles containing insulin-responsive intracellular glucose transporters isolated from 3T3-L1 adipocytes by an improved procedure. 304 18

Insulin regulates glucose transport in muscle and adipose tissue by triggering the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.
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PMID:Characterization of insulin-responsive GLUT4 storage vesicles isolated from 3T3-L1 adipocytes. 1059 43

Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha-treated adipocytes correlated with the regulation of specific glycosyltransferases, including the up-regulation of B4GalT5 and Ggta1 galactosyltransferases and down-regulation of ST3Gal6 sialyltransferase. Knockdown of B4GalT5 down-regulated the terminal di-galactose N-glycans, confirming the involvement of this enzyme in the TNF-alpha-regulated N-glycome. SILAC-based quantitative glycoproteomics of enriched N-glycopeptides with and without deglycosylation were used to identify the protein and glycosylation sites modified with these regulated N-glycans. The combined proteome and glycoproteome workflow provided a relative quantification of changes in protein abundance versus N-glycosylation occupancy versus site-specific N-glycans on a proteome-wide level. This revealed the modulation of N-glycosylation on specific proteins in IR, including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane.
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PMID:Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes. 2653 98