Gluconeogenic enzymes in sheep liver

Abstract

Abstract 1. 1. The subcellular distributions of pyruvate carboxylase and phosphoenol-pyruvate (PEP) carboxykinase were investigated in the livers of normal, fasted and diabetic sheep. The intramitochondrial distributions of these enzymes were determined following digitonin fractionation of isolated mitochondria and the use of marker enzymes. 2. 2. In normal liver all the pyruvate carboxylase and 30% of the PEP carboxykinase were shown to be located in the mitochondrial matrix. The remaining 70% of the PEP carboxykinase activity was found in the cytosol. In fasted and diabetic sheep up to 40% of the total pyruvate carboxylase activity was found in the cytosol, but studies with marker enzymes showed that this was due to release from mitochondria. These results suggested that the mitochondria were more fragile in these physiological conditions. This hypothesis was substantiated by electron micrographs which showed that in liver sections from diabetic sheep a high proportion of mitochondria were several times the normal size, but that in preparations of isolated mitchondria from diabetic sheep liver, there were only normal-sized mitochondria and a large amount of membrane fragments. 3. 3. Further evidence for the common identity of the pyruvate carboxylase of the mitochondrial and cytosol fractions was obtained by the equivalent titration of both enzymes with antibodies prepared to purified mitochondrial pyruvate carboxylase from the livers of normal sheep, and by Ouchterlony double diffusion analyses. 4. 4. It is concluded therefore that although there are marked increases in liver pyruvate carboxylase in fasted and diabetic sheep, the activity is exclusively mitochondrial in vivo under all conditions investigated. PEP carboxykinase activity, however, is distributed between the mitochondria and cytosol with similar increases in activity occuring in both fractions in the diabetic sheep.