Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase were purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (MW 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the 3 substrates. Phosphatases I1 (MW 180,000) and I3 (MW 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the 3 substrates. In contrast, phosphatase I2 (MW 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the MW of all phosphatases changed; protein phosphatases I2, I3 and II were brought to an MW of 35,000, while phosphatase I1 was reduced on an MW of 69,000. Glycogen synthase phosphatase activity was decreased in all 4 phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. THe HMG-CoA reductase phosphatase and phosphorylase phosphatase activites of phosphatases I2, I3 and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The 4 phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3 and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen cynthase phosphatase activity presented less inhibition in the low-MW forms. A comparison was made with other protein phosphatases previously reported in the literature.
