Degradation of phosphoenolpyruvate carboxykinase (guanosine triphosphate)in vivo and in vitro

Abstract

1. Phosphoenolpyruvate carboxykinase (GTP) in the cytosol fraction of liver was labelled in young rats by the injection of [3H]leucine and then isolated with specific antibody. Antibody-antigen precipitates from `pulse'-labelled animals and from animals in which the content of radioactive enzyme had been decreased by a period of degradation were separated by electrophoresis on sodium dodecyl sulphate–polyacrylamide gels. No radioactive breakdown products were found. 2. 3H-labelled phosphoenolpyruvate carboxykinase (GTP) was purified from rat liver and used to measure degradation in vitro. There was first a loss of catalytic activity, then a disappearance of immunological activity and finally a loss of solubility before any evidence of proteolytic cleavage. Proteolytic-cleavage fragments, when found, were also insoluble. 3. An analysis of the subcellular location of enzyme inactivation showed that phosphoenolpyruvate carboxykinase (GTP) was stable when incubated with liver cytosol fraction and was inactivated most rapidly by the microsomal fraction. 4. We propose that denaturation of the enzyme is the rate-limiting step in degradation in vivo, and precedes proteolytic cleavage when the enzyme is incubated with liver preparations in vitro.