Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase

Thomas Soderling, A. K. Srivastava, M. A. Bass, B. S. Khatra

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Skeletal muscle glycogen a4-synthase (EC 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6-P-Sepharose affinity column and then on a phosphocellulose column. This preparation of glycogen synthase was tested as a substrate for purified skeletal muscle phosphorylase kinase (ATP:phosphorylase-b phosphotransferase. EC 2.7.1.38). Phosphorylase kinase (1-10 μg/ml or 0.03-0.3 μM) catalyzes rapid phosphorylation of glycogen synthase (4.5 μM) associated with conversion of the active a form to the less active b form. In the reaction, >95% of the 32P incorporation from [γ-32P]ATP goes into the synthase subunit almost exclusively in the trypsin-insensitive region which is responsible for synthase a-to-b conversion. Synthase phosphorylation or inactivation catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, is ATP dependent, is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased 10-fold by prior activation of the phosphorylase kinase with MgATP and cyclic AMP. With activated phosphorylase kinase at pH 8.2 the apparent K(m) and V(max) are approximately 70μM and 4 μmol/min per mg with glycogen synthase as substrate and 70 μM and 9 μmol/min per mg with phosphorylase as substrate. It is concluded that glycogen synthase is a substrate in vitro for phosphorylase kinase, a Ca2+-dependent enzyme. The possible physiological significance of this reaction is discussed.

Original languageEnglish (US)
Pages (from-to)2536-2540
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume76
Issue number6
StatePublished - 1979
Externally publishedYes

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Phosphorylase Kinase
Glycogen Synthase Kinases
Glycogen Synthase
Phosphorylation
Adenosine Triphosphate
Skeletal Muscle
Phosphorylases
Ethylene Glycol
Phosphoric Monoester Hydrolases
Cyclic AMP
Ether
Sepharose
Trypsin
Chromatography
Phosphotransferases
Acids
Enzymes

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase. / Soderling, Thomas; Srivastava, A. K.; Bass, M. A.; Khatra, B. S.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 76, No. 6, 1979, p. 2536-2540.

Research output: Contribution to journalArticle

Soderling, Thomas ; Srivastava, A. K. ; Bass, M. A. ; Khatra, B. S. / Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase. In: Proceedings of the National Academy of Sciences of the United States of America. 1979 ; Vol. 76, No. 6. pp. 2536-2540.
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abstract = "Skeletal muscle glycogen a4-synthase (EC 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6-P-Sepharose affinity column and then on a phosphocellulose column. This preparation of glycogen synthase was tested as a substrate for purified skeletal muscle phosphorylase kinase (ATP:phosphorylase-b phosphotransferase. EC 2.7.1.38). Phosphorylase kinase (1-10 μg/ml or 0.03-0.3 μM) catalyzes rapid phosphorylation of glycogen synthase (4.5 μM) associated with conversion of the active a form to the less active b form. In the reaction, >95{\%} of the 32P incorporation from [γ-32P]ATP goes into the synthase subunit almost exclusively in the trypsin-insensitive region which is responsible for synthase a-to-b conversion. Synthase phosphorylation or inactivation catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, is ATP dependent, is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased 10-fold by prior activation of the phosphorylase kinase with MgATP and cyclic AMP. With activated phosphorylase kinase at pH 8.2 the apparent K(m) and V(max) are approximately 70μM and 4 μmol/min per mg with glycogen synthase as substrate and 70 μM and 9 μmol/min per mg with phosphorylase as substrate. It is concluded that glycogen synthase is a substrate in vitro for phosphorylase kinase, a Ca2+-dependent enzyme. The possible physiological significance of this reaction is discussed.",
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