Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase

Arch Biochem Biophys. 1993 Dec;307(2):248-52. doi: 10.1006/abbi.1993.1586.


Sucrose-phosphate synthase (SPS; EC is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism*
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Phosphopeptides / chemistry
  • Phosphopeptides / genetics
  • Phosphorylation
  • Phosphoserine / isolation & purification
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Analysis
  • Sequence Homology, Amino Acid
  • Vegetables
  • Zea mays


  • Peptide Fragments
  • Phosphopeptides
  • Recombinant Proteins
  • Phosphoserine
  • Glucosyltransferases
  • sucrose-phosphate synthase