Purification and characterization of a protein phosphatase from rat liver acting on key enzymes of glucose metabolism

A phosphoprotein phosphatase has been purified from rat liver cytosol. The purification involved chromatography on DEAE-cellulose, Sephacryl S-200, fast protein liquid chromatography (FPLC) and sucrose density gradient centrifugation. It resulted in an almost homogeneous enzyme with a relative molecular mass, Mr, of 90000 by gel filtration and sucrose gradient centrifugation and Mr= 44500 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE).Therefore it seems to be a dimeric enzyme. This protein phosphatase (termed PFK-phosphatase) is completely dependent on Mg2+, which can be replaced partly by Mn2+. It can be eluted from DEAE-cellulose with 120 mM NaCl, is not affected by Ca2+, 100μM trifluoperazine or the heat-stable inhibitor-2. Inhibition occurs with phosphate, ammonium sulfate and fluoride. PFK-phosphatase dephosphorylates preferentially the α subunit of phosphorylase kinase (α/β dephosphorylation ratio 5–10). Phosphorylase a, mixed histone and casein do not serve as substrates. The enzyme dephosphorylates effectively the key enzymes of glucose metabolism 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and 6-phosphofructo-2-kinase. Using this protein phosphatase and the catalytic subunit of cAMP-dependent protein kinase, a complete phosphorylation, dephosphorylation and rephosphorylation cycle was possible with 6-phosphofructo-1-kinase as substrate.