A latent form of protein phosphatase 1 α associated with bovine heart myofibrils

Abstract

The catalytic subunit of the major protein phosphatase associated with bovine cardiac myofibrils was purified to homogeneity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme revealed only one band with an apparent molecular weight of 37 000. On gel filtration chromatography, the phosphatase activity and the protein co-eluted as a single peak with an apparent molecular weight of 37 000. The purified enzyme was identified as the catalytic subunit of protein phosphatase 1, as determined by sensitivity to inhibitor 1, inhibitor 2, okadaic acid and by specific immunostaining. Evidence obtained with specific antipeptide antibodies demonstrated that this myofibril protein phosphatase was predominately the α isoform of protein phosphatase 1. The purified catalytic subunit was completely inactive. It was activated by pretreatment with Co 2+/trypsin in the presence of high ionic strength. Treatment with trypsin alone did not activate the latent enzyme. The enzyme was also activated by Co 2+ or Mn 2+ alone but not by Ca 2+, Mg 2+, Ni 2+, Cu 2+ or Zn 2+. Activation of the enzyme was not reversed by removal of Co 2+, but Mn 2+-activated phosphatase activity was partially reversed when Mn 2+ was removed. The catalytic subunit could form a 1:1 complex with inhibitor 2 in vitro. The resulting holoenzyme was also activated by pretreatment with Co 2+. Since phosphatase 1α is the major phosphatase associated with cardiac myofibril, it is suggested that it is responsible for the dephosphorylation of myosin and other myofibril phosphoproteins.