Purification of phosphoenolpyruvate carboxykinase from the cytosol fraction of rat liver and the immunochemical demonstration of differences between this enzyme and the mitochondrial phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) has been isolated and purified from the cytosol fraction of rat liver to give a specific activity of 14 to 16 moles of bicarbonate tied per min per mg of protein. The enzyme is homogeneous on disc electrophoresis and Sephadex G-100 chromatography and has a molecular weight of 74,500 with an isoelectric point of 5.04. Antibodies prepared in rabbits against the pure enzyme show identical titration curves against the cytosol P-enolpyruvate carboxykinase of liver and adipose tissue. A single continuous precipitation line is obtained on Ouchterlony double dausion precipitation analysis when pure enzyme and the cytosol P-enolpyruvate carboxykinase of liver and adipose tissue are compared. On the other hand, the solubilized mitochondrial P-enolpyruvate carboxykinase of rat liver is not precipitated by the antibody to the cytosol enzyme. We conclude that the mitochrondrial and cytosol P-enolpyruvate carboxykinases are distinct enzymes, notwithstanding their uniform kinetic and physical properties.