Identification of ATP citrate lyase as a phosphoprotein

ATP citrate lyase has been purified from rat liver by a new procedure which results in high yields of an intact and stable enzyme. The pure lyase (specific activity approximately equal to 10 at 25 degrees C) exhibits a single protein band upon sodium dodecyl sulfate (SDS)-gel electrophoresis (Mr = 110,000). This procedure minimizes protease degradation that usually occurs when the enzyme is isolated by previously described isolation methods. In addition, the lyase is shown to be a phosphoprotein. 32P-labeled lyase has been purified from rat liver following an intraperitoneal injection of inorganic [32P]phosphate into the animals. It has been demonstrated that this phosphate (structural phosphate) behaves as a serine phosphate and is not the same as the enzyme-bound phosphate (catalytic phosphate) that is derived from ATP during the lyase reaction. There are 2 structural phosphate residues for each enzyme tetramer molecule. Removal of the structural phosphate has been accomplished using a partially purified phosphatase derived from rat liver. The dephospholyase has the same Vmax as the native phosphoenzyme. Evidence indicates that the structural phosphate resides in a protease-sensitive region of the native enzyme.