Functional intracellular glutaminase activity in intact astrocytes

Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the functional intracellular phosphate-activated glutaminase activity was measured by the formation of³H₂O froml-2-³H]glutamine (Anal. Biochem. 127:134–142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of¹⁴CO₂ release froml-1-¹⁴C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of¹⁴CO₂ release froml-1-¹⁴C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 M glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%. The addition of 10 mM phosphate and the alpha keto acids of isoleucine and valine marginally increased intracellular glutaminase activity. The addition of 0.4 mM ammonium chloride to the medium had no effect. An increase in media pH from 6.8 to 7.7 increased intracellular glutaminase activity almost 2 fold. These results provide evidence that phosphate-activated glutaminase activity in vivo is regulated by cellular metabolites, that its functional activity is 5–9% of the rate obtained using extracts, and this functional activity is sufficient to account for the rate of glutamine oxidation.Special issue dedicated to Dr. Elling Kvamme