TY - JOUR
T1 - Substrate specificity of phosphorylase kinase
T2 - Effects of heparin and calcium
AU - Bollen, Mathieu
AU - Kee, Scott M.
AU - Graves, Donald J.
AU - Soderling, Thomas R.
N1 - Funding Information:
i Supported in part by NIH and GM-09587 (D.J.G.). ’ Present address: Afdeiing Universiteit Leuven, Campus traat B-3000, Leuven, Belgium. a To whom correspondence
PY - 1987/5/1
Y1 - 1987/5/1
N2 - 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.
AB - 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.
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U2 - 10.1016/0003-9861(87)90122-6
DO - 10.1016/0003-9861(87)90122-6
M3 - Article
C2 - 3107473
AN - SCOPUS:0023266487
SN - 0003-9861
VL - 254
SP - 437
EP - 447
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -