Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver.
The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004355:Drug Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006005:Phosphorylases; D011817:Rabbits; D013997:Time Factors
D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D006005:Phosphorylases; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium