ISOLATION OF PHOSPHOOLIGOSACCHARIDE PHOSPHOINOSITOL GLYCAN FROM CAVEOLAE AND CYTOSOL OF INSULIN-STIMULATED CELLS

A phosphooligosaccharide has been proposed as a second messenger of in sulin. It is believed to be structurally related to the carbohydrate m oiety of phosphatidylinositol glycan anchors of many cell surface prot eins. Herein we demonstrate that [P-32]phosphate in freshly isolated a dipocytes and [H-3]galactose in cultured hepatoma cells (H4IIE) labele d the same set of three different glycolipids. With all three, the rad iolabel was made water soluble by phosphatidylinositol(glycan)-specifi c phospholipase C or D catalyzed hydrolysis. We isolated the three pho spholipase C-released substances. One of them was susceptible to nitro us acid deamination, indicative of a hexosamine with a free amino grou p. This phosphooligosaccharide structure had an apparent molecular mas s between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and inte rconvertible species. Adipocytes stimulated with insulin accumulated t he nitrous acid sensitive phosphooligosaccharide: after stimulation th e intracellular level of free phosphooligosaccharide increased threefo ld within 5 min, fell off during the next few minutes and then remaine d at a slightly elevated level. After insulin stimulation the intracel lular concentration of free phosphooligosaccharide was >1,000-fold hig her than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insul inomimetic effects on protein phosphorylation of insulin target protei ns in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [P-32]phosphooligosaccharide of the plasma membrane w as found to be localized in plasma membrane domains apparently corresp onding to caveolae. Lipid-bound [P-32]phosphooligosaccharide was found also in the microsomal fraction.