Modulation of rat liver hydroxymethylglutaryl-CoA reductase by protein phosphatases: Purification of nonspecific hydroxymethylglutaryl-CoA reductase phosphatases

Abstract

Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been 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 ( M r 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I 1 ( M r 180,000) and I 3 ( M r 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I 2 ( M r 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 M r of all phosphatases changed; protein phosphatases I 2, I 3, and II were brought to an M r of 35,000, while phosphatase I 1 was reduced to an M r of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I 1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I 2, I 3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn 2+ and Mg 2+. In general, Mn 2+ was a better activator than Mg 2+, and phosphatase I 1 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 I 1, I 3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low- M r forms. A comparison has been made with other protein phosphatases previously reported in the literature.