, Volume 223, Issue 3, pp 468–478 | Cite as

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

  • Guang-Huar Young
  • Han-Min Chen
  • Chi-Tsai Lin
  • Kuang-Ching Tseng
  • Jiann-Shing Wu
  • Rong-Huay JuangEmail author
Original Article


A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.


Ipomoea Protein kinase Protein phosphorylation Sweet potato roots Starch phosphorylase 



Calcium-dependent protein kinase


Glucose 1-phosphate


Glycogen phosphorylase


High-affinity type SP (Pho2)


Low-affinity type SP (Pho1)


myelin basic protein


Polyvinylidene fluoride


Starch phosphorylase



We thank Drs. D.S. Bendall and B.G. Schlarb-Ridley (University of Cambridge); Dr. S.C. Chang (Harvard Medical School); Drs. Y.Y. Charng and C.S. Chen (Academia Sinica, ROC); Dr. C.M. Chiang (Case Western Reserve University); Drs. P.L. Huang, A.Y. Wang, and C.C. Yang (National Taiwan University); and Dr. A.B. Tobin (University of Leicester) for their helpful suggestions and discussions. We also thank Dr. J. Hook for reading the article. Part of this work was supported by the National Science Council, ROC.


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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Guang-Huar Young
    • 1
  • Han-Min Chen
    • 2
  • Chi-Tsai Lin
    • 3
  • Kuang-Ching Tseng
    • 1
  • Jiann-Shing Wu
    • 1
  • Rong-Huay Juang
    • 1
    Email author
  1. 1.Department of Biochemical Science and Technology, Institute of Microbiology and BiochemistryNational Taiwan UniversityTaipeiTaiwan
  2. 2.Department of Life ScienceCatholic Fu-Jen UniversityTaipeiTaiwan
  3. 3.Institute of Bioscience and BiotechnologyNational Taiwan Ocean UniversityKeelungTaiwan

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