Glycogenin (ON) is a sclf-glucosylating protein involved in the initiation of the synthesis of glycogen molecule. Stable clones of Rat-1 fibroblasts overexpressing rabbit muscle GN were generated. Detection of ON by Western Mot analysis required either preliminary incubation of the cells in a glucose-depleted medium or treatment of the cell extract with a-amylase. Immunofluorescence experiments showed diffused distribution of GN suggestive of a cytosolic localization of the protein. Expession of GN did not change glycogen and glycogen synthase (GS) levels in Rat-1 fibroblasts. However, the presence of GN caused a significant redistribution of glycogen and GS from the low speed pellet to the soluble fraction of the cell homogenates. Overexpression of GN increased the -/+glucose-6-P activity ratio of GS in cells incubated in low glucose media. Analysis of [35S]methionine- or [14C]glucose-labeled proteins in stable clones by 2D-gel electrophorcsis revealed a continuum of GN-containing species with Mr from 38 IcDa to more than 400 kDa. Larger species were more positively charged than smaller molecules. Incubation of Rat-1 clones with [32P]phosphate followed by immunoprecipitation showed a low level of phosphorylation of GN, the significance of which is not clear. These results indicate new notentional mechanisms controling glycogen biogenesis.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology