Regulation of Renal Ammoniagenesis

Abstract

Abstract Rat kidney contains two distinct glutaminase isoenzymes; one of which is phosphate-dependent, the other is phosphate-independent but is strongly activated by maleate. The phosphate-dependent glutaminase is contained within mitochondria. Its activity cofractionates with mitochondrial marker activities during differential centrifugation and on sucrose gradients using both isopycnic and sedimentation velocity techniques. Incubation of mitochondria with borate buffer results in increased phosphate-dependent glutaminase, cytochrome oxidase, and malate dehydrogenase activities, indicating that this glutaminase activity may be latent. Submitochondrial fractionation by digitonin-Lubrol treatment in the presence of borate indicated that this glutaminase isoenzyme is contained in the inner mitochondrial membrane. This conclusion was confirmed by a swell-shrink, sonication procedure carried out in the absence of borate. Only 25 to 35 % of the phosphate-independent glutaminase was associated with the mitochondrial fraction obtained by differential centrifugation; the remaining activity was pelleted by subsequent centrifugation at 40,000 x g for 30 min (heavy microsomes). The phosphate-independent glutaminase in both fractions banded during isopycnic centrifugation with a mean density (1.166) similar to that of light mitochondria. However, the results of sedimentation velocity centrifugation studies showed that this isoenzyme was contained in an organelle distinct from mitochondria. The phosphate-independent glutaminase did not fractionate with marker activities for lysosomes, microsomes, or plasma membranes during differential centrifugation or isopycnic gradient analysis and may be contained in some as yet unidentified organelle.