Regulation of protein phosphatase‐1 G from rabbit skeletal muscle

Abstract

The glycogen‐associated form of protein phosphatase‐1 (PP‐1 G ) 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‐1 G 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‐1 G is 5–8 times higher than that of released C subunit or unbound PP‐1 G , which are strongly inhibited at these ionic strengths. The activity of glycogen‐bound PP‐1 G 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‐1 G activity is only enhanced compared to free C subunit at near physiological ionic strength and when both PP‐1 G 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 V max . From the determined specificity constant, k ′ cat / K ′ m ∼ 4 × 10 6 s −1 M −1 , it was calculated that at physiological levels of glycogen‐bound PP‐1 G (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‐1 G toward the glycogen‐metabolising enzymes at physiological ionic strength compared to free C subunit substantiates the role of PP‐1 G 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.