The glycogen-associated form of protein phosphatase-1 (PP-1G) comprises a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit. In the preceding paper in this issue of the journal we showed that the C subunit is released from PP-1G in response to phosphorylation of the G subunit by cAMP-dependent protein kinase. We now show that at 0.15–02 M KCl the phosphorylase phosphatase activity of glycogen-bound PP-1G is 5–8 times higher than that of released C subunit or unbound PP-1G, which are strongly inhibited at these ionic strengths. The activity of glycogen-bound PP-1G towards glycogen synthase was about 5-fold higher than that of released C subunit at 0.15M KCl. Studies with glycogen-bound substrates and myosin P-light chain (which does not interact with glycogen) indicated that PP-1G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP-1G and substrate are glycogen-associated.
The inhibition by increasing ionic strength and enhanced activity upon binding to glycogen reflected changes in K′m, but not Vmax. From the determined specificity constant, k′cat/K′m∼ 4 × 106 s−1 M−1, it was calculated that at physiological levels of glycogen-bound PP-1G (200 nM) and phosphorylase (70 μM). dephosphorylation of the latter could occur with a half time of 15 s, sufficient to account for inactivation rates in vivo.
The much higher catalytic efficiency of glycogen-bound PP-1G toward the glycogen-metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP-1G in the regulation of these substrates, and establishes a novel mechanism for selectively regulating their phosphorylation states in response to adrenalin and other factors affecting phosphorylation of the G subunit.
glycogen-associated form of protein phosphatase-1
catalytic subunit of protein phosphatase-1
cAMP-dependent protein kinase
apparent Michaelis constant
Protein phosphatase (EC 184.108.40.206)
protein kinase (EC 220.127.116.11)
phosphorylase kinase (EC 18.104.22.168)
glycogen synthase (EC 22.214.171.124)
glycogen phosphorylase (EC 126.96.36.199)