Further studies on the structure of the glycogen‐bound form of protein phosphatase‐1 from rabbit skeletal muscle

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Abstract
We have reported previously that the glycogen‐bound form of protein phosphatase‐1 (termed PP‐1G) is a heterodimer, composed of the 37‐kDa catalytic (C) subunit complexed to a 103‐kDa G‐subunit that anchors the enzyme to glycogen [Strålfors, P., Hiraga, A. and Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. An antibody raised against a synthetic peptide corresponding to the phosphorylation site on the G‐subunit was found to immunoprecipitate PP‐1G specifically. No precipitation occurred if the antibody was preincubated with the synthetic peptide, or if PP‐1G was replaced by either the isolated C‐subunit or protein phosphatase‐2A. The results confirm by a new and independent method that the G‐subunit is complexed to the C‐subunit, and that it is not a contaminant. The G‐subunit is remarkably sensitive to proteolysis. At the final stage of purification, PP‐1G was eluted as a broad peak of activity. The leading fractions contained the 37‐kDa C‐subunit and 103‐kDa G‐subunit, while the central and trailing fractions comprised the 37‐kDa C‐subunit plus a number of bands with molecular masses ranging over 40–80 kDa. The 40–80‐kDa bands were phosphorylated by cyclic‐AMP‐dependent protein kinase and tryptic digestion generated the identical phosphopeptides obtained by trypsinisation of the 103‐kDa G‐subunit. Furthermore, antibody to the G‐subunit immunoprecipitated protein phosphatase activity quantitatively in the leading, central and trailing fractions. The results demonstrate that the 40–80‐kDa polypeptides are fragments of the G‐subunit, and that fragments as small as 40 kDa retain the sites of phosphorylation as well as the ability to interact with the C‐subunit and with glycogen. Khatra [J. Biol. Chem. (1986) 261, 8944–8952] reported that the glycogen‐bound form of protein phosphatase‐1 did not contain a G‐subunit and that it was a dimer composed of two identical C‐subunits. The present work has shown that this proposal is incorrect, and that proteolysis of the G‐subunit to fragments that stain very poorly with Coomassie blue can explain why this error was made.