Synthase phosphatase, phosphorylase phosphatase and histone phosphatase in rat liver were measured using as substrate purified liver synthase D, phosphorylase a and 32P-labelled phosphorylated f1 histone, respectively. The three phosphatase enzymes had different sedimentation characteristics. Both synthase phosphatase and phosphorylase phosphatase were found to sediment with the microsomal fraction under our experimental conditions. Only 10% of histone phosphatase was in this fraction; the majority was in the cytosol. No change in histone phosphatase was observed in the adrenalectomized fasted rat whereas synthase phosphatase and phosphorylase phosphatase activities were decreased 5-10-fold. Fractionation of liver extract with ethanol produced a dissociation of the three phosphatase activities. When a partially purified fraction was put on a DEAE-cellulose column, synthase phosphatase and phosphorylase phosphatase both exhibited broad elution profiles but their activity peaks did not coincide. Histone phosphatase eluted as a single discrete peak. When the supernatant of CaCl2-treated microsomal fraction was put on a Sepharose 4B column, the majority of synthase phosphatase was found to elute with the larger molecular weight proteins whereas the majority of phosphorylase phosphatase eluted with the smaller species. Histone phosphatase migrated as a single peak and was of intermediate size. Synthase phosphatase was inhibited by phosphorylase a (Ki < 1 unit/ml) and phosphorylase phosphatase by synthase D (K1 ≈ units/ml). The inhibition of synthase phosphatase by phosphorylase a was kinetically non-competitive with substrate. Histone phosphatase activity was not inhibited by synthase D or by phosphorylase a. The above results suggest that different proteins are involved in the dephosphorylation of synthase D, phosphorylase a and histone in the cell.
Bibliographical noteFunding Information:
This work was supported in part by a grant from the American Diabetes Association, Minnesota Affiliate. The authors gratefully acknowledge the excellent technical assistance of Deborah Pahl in these studies.