The protein phosphatases involved in cellular regulation: Purification and characterisation of the glycogen‐bound form of protein phosphatase‐1 from rabbit skeletal muscle

A type‐1 protein phosphatase (protein phosphatase‐1_G) was purified to homogeneity from the glycogenprotein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase‐1_G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel‐filtration on Sephadex G‐100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37‐kDa type‐1 protein phosphatase catalytic subunit (protein phosphatase‐1_C) isolated from ethanol‐treated muscle extracts, as judged by peptide mapping. The G component was the glycogen‐binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels. Protein phosphatase‐1_G was inhibited by inhibitor‐1 and inhibitor‐2, but unlike protein phosphatase‐1_c, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase‐1_G was inactivated very rapidly by inhibitor‐1. Protein phosphatase‐1_G interacted with inhibitor‐2 (1–2) to form an inactive species, with the structure GCI‐2. This form could be activated by preincubation with Mg‐ATP and glycogen synthase kinase‐3. The G subunit could be phosphorylated on a serine residue(s) by cyclic‐AMP‐dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase‐3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase‐1_G by inhibitor‐1. The relationship of the G subunit to the ‘deinhibitor protein’ is discussed.