Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Jun 1;268(2):401–407. doi: 10.1042/bj2680401

Purification, characterization and partial amino acid sequence of glycogen synthase from Saccharomyces cerevisiae.

A Carabaza 1, J Arino 1, J W Fox 1, C Villar-Palasi 1, J J Guinovart 1
PMCID: PMC1131446  PMID: 2114092

Abstract

Glycogen synthase from Saccharomyces cerevisiae was purified to homogeneity. The enzyme showed a subunit molecular mass of 80 kDa. The holoenzyme appears to be a tetramer. Antibodies developed against purified yeast glycogen synthase inactivated the enzyme in yeast extracts and allowed the detection of the protein in Western blots. Amino acid analysis showed that the enzyme is very rich in glutamate and/or glutamine residues. The N-terminal sequence (11 amino acid residues) was determined. In addition, selected tryptic-digest peptides were purified by reverse-phase h.p.l.c. and submitted to gas-phase sequencing. Up to eight sequences (79 amino acid residues) could be aligned with the human muscle enzyme sequence. Levels of identity range between 37 and 100%, indicating that, although human and yeast glycogen synthases probably share some conserved regions, significant differences in their primary structure should be expected.

Full text

PDF
401

Images in this article

403Fig. 1.
on p.403
403Fig. 2.
on p.403
404Fig. 3.
on p.404

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Browner M. F., Nakano K., Bang A. G., Fletterick R. J. Human muscle glycogen synthase cDNA sequence: a negatively charged protein with an asymmetric charge distribution. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1443–1447. doi: 10.1073/pnas.86.5.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Camici M., DePaoli-Roach A. A., Roach P. J. Rabbit liver glycogen synthase. Purification and comparison of the properties of glucose-6-P-dependent and glucose-6-P-independent forms of the enzyme. J Biol Chem. 1984 Mar 25;259(6):3429–3434. [PubMed] [Google Scholar]
  4. Flotow H., Roach P. J. Synergistic phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase and casein kinase I. Implications for hormonal regulation of glycogen synthase. J Biol Chem. 1989 Jun 5;264(16):9126–9128. [PubMed] [Google Scholar]
  5. Gilboe D. P., Larson K. L., Nuttall F. Q. Radioactive method for the assay of glycogen phosphorylases. Anal Biochem. 1972 May;47(1):20–27. doi: 10.1016/0003-2697(72)90274-6. [DOI] [PubMed] [Google Scholar]
  6. Huang K. P., Cabib E. Yeast glycogen synthetase in the glucose 6-phosphate-dependent form. I. Purification and properties. J Biol Chem. 1974 Jun 25;249(12):3851–3857. [PubMed] [Google Scholar]
  7. Jaspers S. R., Rulfs J., Johnson G. L., Mole J. E., Miller T. B., Jr Amino-terminal sequence analysis of rat heart and muscle glycogen synthase: homology to the rabbit enzyme and the implications for hormonal control. Arch Biochem Biophys. 1989 Feb 1;268(2):630–636. doi: 10.1016/0003-9861(89)90330-5. [DOI] [PubMed] [Google Scholar]
  8. Jett M. F., Soderling T. R. Purification and phosphorylation of rat liver glycogen synthase. J Biol Chem. 1979 Jul 25;254(14):6739–6745. [PubMed] [Google Scholar]
  9. Kaslow H. R., Lesikar D. D., Antwi D., Tan A. W. L-type glycogen synthase. Tissue distribution and electrophoretic mobility. J Biol Chem. 1985 Aug 25;260(18):9953–9956. [PubMed] [Google Scholar]
  10. Kaslow H. R., Lesikar D. D. Isozymes of glycogen synthase. FEBS Lett. 1984 Jul 9;172(2):294–298. doi: 10.1016/0014-5793(84)81144-8. [DOI] [PubMed] [Google Scholar]
  11. Kumar A., Larsen C. E., Preiss J. Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose:alpha-1,4-glucan, 4-glucosyltransferase as deduced from the nucleotide sequence of the glgA gene. J Biol Chem. 1986 Dec 5;261(34):16256–16259. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Mahrenholz A. M., Wang Y. H., Roach P. J. Catalytic site of rabbit glycogen synthase isozymes. Identification of an active site lysine close to the amino terminus of the subunit. J Biol Chem. 1988 Aug 5;263(22):10561–10567. [PubMed] [Google Scholar]
  14. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  15. Miller R. E., Miller E. A., Fredholm B., Yellin J. B., Eichner R. D., Mayer S. E., Steinberg D. Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. II. Glycogen synthase. Biochemistry. 1975 Jun 3;14(11):2481–2488. doi: 10.1021/bi00682a030. [DOI] [PubMed] [Google Scholar]
  16. Poulter L., Ang S. G., Gibson B. W., Williams D. H., Holmes C. F., Caudwell F. B., Pitcher J., Cohen P. Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry. Eur J Biochem. 1988 Aug 15;175(3):497–510. doi: 10.1111/j.1432-1033.1988.tb14222.x. [DOI] [PubMed] [Google Scholar]
  17. Rothman-Denes L. B., Cabib E. Glucose 6-phosphate dependent and independent forms of yeast glycogen synthetase. Their properties and interconversions. Biochemistry. 1971 Mar 30;10(7):1236–1242. doi: 10.1021/bi00783a021. [DOI] [PubMed] [Google Scholar]
  18. Thomas J. A., Schlender K. K., Larner J. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal Biochem. 1968 Oct 24;25(1):486–499. doi: 10.1016/0003-2697(68)90127-9. [DOI] [PubMed] [Google Scholar]
  19. Wang Y. H., Bell A. W., Hermodson M. A., Roach P. J. Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase. J Biol Chem. 1986 Dec 25;261(36):16909–16915. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES