Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.
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 [32]phosphate in freshly isolated adipocytes and [3H]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 [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction.
Parpal
S
S
Department of Cell Biology, Faculty of Health Sciences, University of Linköping, Sweden.
Gustavsson
J
J
Strålfors
P
P
D017667:Adipocytes / Q000737:chemistry / Q000187:drug effects; D000818:Animals; D001345:Autoradiography; D006528:Carcinoma, Hepatocellular; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry*; D005690:Galactose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D009844:Oligosaccharides / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D010710:Phosphates / Q000302:isolation & purification / Q000378:metabolism; D010716:Phosphatidylinositols / Q000302:isolation & purification* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011134:Polysaccharides / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000737:chemistry; D014316:Tritium
D007328:Insulin; D009844:Oligosaccharides; D010710:Phosphates; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D011134:Polysaccharides; D014316:Tritium; D005690:Galactose