Liver protein phosphatases: Studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000

Abstract

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M r 35,000. The M r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M r 35,000 form by treatment with either ethanol or urea. Treatment of the M r 260,000 form with trypsin led to its conversion to M r 225,000 and a M r 35,000 form. A minor cytosolic form of M r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.