Substrate specificity of phosphorylase kinase: Effects of heparin and calcium

Mathieu Bollen, Scott M. Kee, Donald J. Graves, Thomas R. Soderling

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Phosphorylase b and two peptides with sequences homologous to phosphorylation site 2 (syntide 2) and site 3 (syntide 3) of glycogen synthase were compared as substrates for purified muscle phosphorylase kinase. The substrate specificity of phosphorylase kinase varied according to whether heparin (at pH 6.5) or Ca2+ (at pH 8.2) was used as a stimulator of its activity. Phosphorylase b was preferentially phosphorylated in the presence of Ca2+; the rate of syntide 2 phosphorylation was the same for both stimulators; and the phosphorylation of syntide 3 was completely dependent on the presence of heparin. A kinetic analysis confirmed this stimulator-dependent substrate specificity since both the Vmax and Km for these substrates were affected diversely by heparin and Ca2+. Heparin stimulated phosphorylase kinase maximally at pH 6.5, whereas the effect of Ca2+ was optimal at a pH above 8. However, the stimulator-related substrate specificity could not be explained by the different pH values at which the effects of the stimulators were assessed. Nor did substrate-directed effects by heparin or Ca2+ apparently play a role. No indications were found for a stimulator-dependent specificity in the phosphorylation of sites in protein substrates of phosphorylase kinase (phosphorylase b, the α-and β-subunits of phosphorylase kinase, or glycogen synthase). The diverse substrate specificity of the calcium- and heparin-dependent activities of phosphorylase kinase could be explained in two ways: either by the existence of separate calcium- and heparinstimulated catalytic sites, or by just one catalytic site with two active conformations. The second possibility is favored by the observation that both calcium and heparin stimulated the isolated γ-subunit (γ · calmodulin complex) of phosphorylase kinase.

Original languageEnglish (US)
Pages (from-to)437-447
Number of pages11
JournalArchives of Biochemistry and Biophysics
Issue number2
StatePublished - May 1 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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