The glycogen‐bound form of protein phosphatase‐1 (PP‐1_g) was previously purified as a heterodimer composed of a 37‐kDa catalytic (C) subunit and a proteolytically sensitive 103‐kDa glycogen‐binding (G) subunit [Stråhlfors, P., Hiraga, A. & Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. In this paper we demonstrate by a variety of criteria that the intact G subunit is a 161‐kDa protein, and that the 103‐kDa species (now termed G′) is itself a product of proteolysis.

A second phosphorylation site for cAMP‐dependent protein kinase (termed site 2) was identified on the G subunit. The site 2 serine was phosphorylated at a comparable rate to site 1, and near stoichiometric phosphorylation could be achieved in the presence and absence of glycogen. Site 2 was dephosphorylated by PP‐1 at a slow rate, whereas site 1 was resistant to autodephosphorylation.

PP‐1_G, as well as the proteolytic activity responsible for degradation of the G subunit, remained tightly associated with glycogen‐protein particles during washing with a variety of solvents. The PP‐1_G holoenzyme was released from glycogen‐protein particles by dilution, with a dissociation half point corresponding to about 10 nM PP‐1_G. Binding experiments with purified PP‐1_G. Binding was not significantly affected by increasing ionic strength to 0.5 M or variation of pH from 6 to 8. The results are consistent with a high‐affinity glycogen‐binding domain on the G subunit, and indicate that at physiological concentrations of phosphatase and glycogen, PP‐1_G should be almost entirely bound to glycogen.