Glycogen Synthetase-D Phosphatase: I. SOME NEW PROPERTIES OF THE PARTIALLY PURIFIED ENZYME FROM RABBIT SKELETAL MUSCLE

Abstract

Abstract Purification of the phospho-protein phosphatase of skeletal muscle which promotes the conversion of the phospho- (D or b) form to the dephospho- (I or a) form of glycogen synthetase (UDPG:glycogen α-4-glucosyltransferase, EC 2.4.I.II) was aided by the use of buffers which include manganese chloride. The Mn2+ (5 mm) appeared to stabilize the enzyme and to insure greater recovery from DEAE-cellulose chromatography than reported previously. The enzyme was purified more than 1,000-fold from the 78,000 x g supernatant of rabbit muscle homogenate. It was essentially free of phosphorylase and synthetase. Phosphatase activity was measured by two methods: (a) as the rate of conversion of glycogen synthetase-D to the I form (D to I conversion) and (b) as the rate of orthophosphate release from 32P-labeled synthetase-D (32Pi release). Inhibition of the phosphatase reaction (more than 50%) was found in the presence of F- (10 mm), Na2SO3 (1 mm), and Pi or PPi (0.2 mm). More than a 2-fold increase in phosphatase activity was found in the presence of divalent metal cations: Mn2+ g Ca2+ g Mg2+ (half-maximal concentration was 0.6 to 1.2 mm). Glucose-6-P (up to 1 mm) had no effect on the rate of 32Pi release, but increased about 2-fold the rate of D to I conversion (at 0.1 mm), as did galactose-6-P and glucosamine-6-P to a lesser extent. A glycogen concentration of 0.5 to 1.5 mg per ml was found to be optimal compared to the slower reaction rates found with greater or lesser concentrations. A histone phosphatase activity found in this preparation, measured using 32P-labeled histone phosphate as substrate, had properties similar to and was copurified with glycogen synthetase-D phosphatase. However, the above carbohydrate-type effectors of synthetase and synthetase-D phosphatase reactions were without effect on the rate of histone dephosphorylation. These findings suggest that these two phosphatase activities may reside in the same enzyme protein.