Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.

• DOI: 10.1083/jcb.131.1.125
• Publication Date: 2004-05-15T01:23:47Z
• Authors: S Parpal; J Gustavsson; P Strålfors
• Keywords: Cell fractionation

A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related the carbohydrate moiety phosphatidylinositol glycan anchors many cell surface proteins. Herein we demonstrate that [32]phosphate in freshly isolated adipocytes and [3H]galactose cultured hepatoma cells (H4IIE) labeled same set three different glycolipids. With all three, radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We C-released substances. One them susceptible nitrous acid deamination, indicative hexosamine with free amino group. This structure had an apparent molecular mass between tetra- pentaglucose gel filtration. By anion-exchange chromatography it separated into two differently charged interconvertible species. Adipocytes stimulated insulin accumulated sensitive phosphooligosaccharide: after stimulation intracellular level increased threefold within 5 min, fell off during next few minutes then remained at slightly elevated level. After concentration > 1,000-fold higher than incubation medium. When prepared from rat livers on preparative scale, oligosaccharide also found exhibit insulinomimetic effects protein phosphorylation target proteins intact adipocytes. subcellular fractionation lipid-bound [32P]phosphooligosaccharide plasma membrane localized domains apparently corresponding caveolae. Lipid-bound microsomal fraction.