Glycogen synthesis in RAT-1 fibroblasts expressing rabbit muscle glycogenin

Alexander Skurat, A. D. Dietrich, Peter Roach

Research output: Contribution to journalArticle

Abstract

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 languageEnglish
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996

Fingerprint

glycogen (starch) synthase
Fibroblasts
Glycogen
Glycogen Synthase
glycogen
fibroblasts
Muscle
rabbits
Rabbits
Rats
Glucose
Muscles
muscles
glucose
synthesis
Clone Cells
clones
rats
Cells
cells

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Glycogen synthesis in RAT-1 fibroblasts expressing rabbit muscle glycogenin. / Skurat, Alexander; Dietrich, A. D.; Roach, Peter.

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

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abstract = "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.",
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AB - 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.

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