Studies on the mechanism by which hepatic citrate synthase activity increases in vitamin B₁₂ deprivation

Hepatic citrate synthase activity has been shown to be increased 2 to 3 fold in vitamin B₁₂ deficiency. Immunochemical titrations of the affinity chromatography purified enzyme obtained from liver of animals with B₁₂ deprivation demonstrated that this increase in activity was the result of a true increase in enzyme protein content. When fixed ratios of aliquots of normal and B₁₂ deprived rat liver homogenates were mixed, the activity measured showed no change from the expected total citrate synthase activity based on the admixture ratios. Partial purification of the enzyme resulted in the expected recovery of the enzyme at each of the purification steps. Thus, it is unlikely that the change in enzyme activity in B₁₂ deprivation was due to the presence of a soluble or easily dissociable normally occurring activator or inhibitor. Ouchterlony double diffusion studies, immunochemical titration, and determination of K(m) values for oxalacetate and acetyl CoA (substrates for citrate synthase) and K(i) values for ATP (inhibitor of citrate synthase) all indicated that the enzyme from the B₁₂ deprived livers was structurally the same as that from normal liver. Hepatic citrate synthase degradation rate constants were shown to be essentially unchanged in B₁₂ deficiency. The rate of hepatic citrate synthase synthesis, under steady state conditions, was shown to be 2.8 fold greater in the B₁₂ deficient animal than in the normal animal. The increased rate of synthesis appeared to explain the increased enzyme content. Finally, no change in specific activity of the enzyme was seen in brain, heart, or kidney in the B₁₂ deprived animal.