Purification and subunit structure of a high-molecular-weight phosphoprotein phosphatase (phosphatase II) from rat liver

Abstract

1. Phosphatase II is a form of phosphoprotein phosphatase originally found in rat liver extract; it has a molecular weight of 160 000 by gel filtration and is highly active towards phosphorylase alpha. This phosphatase has been purified 1800-fold by using DEAE-cellulos (DE-52), aminohexyl--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200 chromatography. Throughout the purification steps, the original molecular weight and substrate specificity of phosphatase II were almost perfectly preserved. 2. The product of the final purification step migrated predominantly as a single protein band on non-denaturing gel electrophoresis. Sodium dodecyl sulfate gel electorphoresis revealed that the enzyme contains two types of subunit, alpha and beta, with molecular weights of 35 000 and 69 000, respectively. When treated with 0.2 M 2-mercaptoethanol at -20 degrees C, phosphatase II was dissociated to release the catalytically active alpha subunit. The beta subunit may be catalytically inactive but interacts with the alpha subunit so that phosphatase II becomes much less susceptible than the alpha subunit to inactivation by ATP or pyrophosphate.