Hepatic L-type pyruvate kinase: separation of unphosphorylated, phosphorylated and proteolytically modified in vivo...

After partial chromatographic purification of rat liver cell sap on DEAE-cellulose, including the removal of type M2 pyruvate kinase, different forms of type L pyruvate kinase were separated by chromatofocusing. Three fractions of pyruvate kinase activity were found, eluting at pH 5.0, 5.2, and 5.3, respectively. The first one was identified as phosphorylated and the second one as unphosphorylated pyruvate kinase. There were strong indications that the third fraction represented a proteolytically modified form of the enzyme, since it co-migrated with a form modified in vitro and had a similarly increased apparent Km for phosphoenolpyruvate. To rule out the possibility of this being a phosphorylated form of pyruvate kinase, the enzyme was incubated with a phosphoprotein phosphatase and then phosphorylated with cAMP-dependent protein kinase. The enzyme was not phosphorylated, like pyruvate kinase modified with subtilisin or calcium-activated protease. There is some evidence that a proteolytically modified pyruvate kinase exists in vivo. This enzyme form has not previously been demonstrated in cell sap, prior to exposure to proteolytic enzymes. The relative amounts of the three forms were determined in livers from starved rats and rats fed on a normal or a carbohydrate-rich diet.