Hormonal control of glycogen synthase in rat hemidiaphragms. Effects of insulin and epinephrine on the distribution of phosphate between two cyanogen bromide fragments

J. C. Lawrence, J. F. Hiken, Anna De Paoli-Roach, Peter Roach

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Abstract

The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (M(app) = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 μM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The latter fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P]phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme.

Original languageEnglish (US)
Pages (from-to)10710-10719
Number of pages10
JournalJournal of Biological Chemistry
Volume258
Issue number17
StatePublished - 1983
Externally publishedYes

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Cyanogen Bromide
Glycogen Synthase
Epinephrine
Rats
Phosphates
Insulin
Phosphorylation
Enzymes
Muscle
Skeletal Muscle
Rabbits
Radioactivity
Electrophoresis
Application programs
Sodium Dodecyl Sulfate
Protein Kinases
Purification
Molecular Weight
Molecular weight
Hormones

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Hormonal control of glycogen synthase in rat hemidiaphragms. Effects of insulin and epinephrine on the distribution of phosphate between two cyanogen bromide fragments",
abstract = "The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (M(app) = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 μM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50{\%}. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The latter fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P]phosphate present in Fragments I and II by about 40{\%}. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme.",
author = "Lawrence, {J. C.} and Hiken, {J. F.} and {De Paoli-Roach}, Anna and Peter Roach",
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T1 - Hormonal control of glycogen synthase in rat hemidiaphragms. Effects of insulin and epinephrine on the distribution of phosphate between two cyanogen bromide fragments

AU - Lawrence, J. C.

AU - Hiken, J. F.

AU - De Paoli-Roach, Anna

AU - Roach, Peter

PY - 1983

Y1 - 1983

N2 - The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (M(app) = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 μM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The latter fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P]phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme.

AB - The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (M(app) = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 μM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The latter fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P]phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme.

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