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| ISOLATION OF PHOSPHOOLIGOSACCHARIDE PHOSPHOINOSITOL GLYCAN FROM CAVEOLAE AND CYTOSOL OF INSULIN-STIMULATED CELLS |
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| Author(s): PARPAL S, GUSTAVSSON J, STRALFORS P |
| Source: JOURNAL OF CELL BIOLOGY Volume: 131 Issue: 1 Pages: 125-135 Published: OCT 1995 |
| Times Cited: 32 References: 41 |
| Abstract: A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that [P-32]phosphate in freshly isolated adipocytes and [H-3]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was >1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [P-32]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [P-32]phosphooligosaccharide was found also in the microsomal fraction. |
| Document Type: Article |
| Language: English |
Addresses:
1. LINKOPING UNIV, FAC HLTH SCI, DEPT CELL BIOL, S-58185 LINKOPING, SWEDEN |
| Publisher: ROCKEFELLER UNIV PRESS, 222 E 70TH STREET, NEW YORK, NY 10021 |
| Subject Category: Cell Biology |
| IDS Number: RX837 |
| ISSN: 0021-9525 |
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