Purification and Immunological Characterization of Mouse Hepatic Phosphoenolpyruvate Carboxykinase

Abstract

The inhibition of glucocorticoid induction and tryptophan activation of phosphoenolpyruvate carboxyfcinase (PEPCK) by bacterial endotoxin may be explained either by decreased synthesis or by inactivation of the enzyme. To differentiate between the two possibilities, mouse hepatic PEPCK was purified using a modification of a method used for securing the enzyme from livers of other species. The techniques included high speed centrifugation of whole liver homogenates, ammonium sulfate fractionation, Sephadex G-100 filtration, DEAE-cellulose ion exchange, hydroxylapatite chromatography, and isoelectric focusing. Antibodies prepared against the purified enzyme gave a single precipitin line in gel diffusion and in Immunoelectrophoresis. The antibodies were used for enzyme titrations in whole liver homogenates of normal and endotoxin poisoned mice, as reported elsewhere.