Glutamine Oxidation by Dissociated Cells and Homogenates of Rat Brain: Kinetics and Inhibitor Studies

Abstract

The rates of [U-¹⁴C]glutamine oxidation to ¹⁴CO² were determined under a variety of experimental conditions using whole homogenates and dissociated cells from rat brain. The pattern of glutamine oxidation by homogenates differed from that by dissociated brain cells in several respects. The rates of glutamine oxidation by dissociated brain cells showed saturation kinetics with an apparent K_m of 0.30 mM. Lineweaver-Burk plots of glutamine oxidation by homogenates revealed two linear segments with two apparent K_m values (0.58 mM and 3.0 mM). In the presence of aminooxyacetate, however, the Lineweaver-Burk plots for homogenates were linear with a single K_m of 0.47 mM. The oxidation of glutamine by homogenates was inhibited by both rotenone and anti-mycin A (80-85%), as were glutamate and glucose oxidation, suggesting that a significant amount of glutamine is oxidized via the tricarboxylic acid cycle. In the presence of aminooxyacetate, glutamine oxidation was inhibited <40%, whereas the oxidation of glutamate was inhibited 75%; in contrast, glucose oxidation was enhanced 50%. The rates of glutamine oxidation by homogenates were highest in the presence of high levels of potassium (50 mM) and low levels of sodium (2.5 mM). Varying ionic composition, however, had little or no effect on the rates of glutamine oxidation by dissociated brain cells. Measurements of glutamine oxidation by homogenates prepared from 2-, 10-, 15-, 25-, and 90-day-old rats revealed little or no age-dependent difference. In contrast, the oxidation by dissociated brain cells from 2-day-old animals was significantly less than that obtained for animals 10 days or older (7.76 vs. 15.6 nmol/h/mg). These results suggest that a significant amount of glutamine can be oxidized via the tricarboxylic acid cycle and is a potential source of energy for the brain. The observed differences between the rates of glutamine oxidation by whole homogenates and dissociated brain cells suggest that the presence of an intact cell membrane may play a role in the regulation of glutamine oxidation. Alternatively, the results may reflect a difference in the concentration of effector(s) or intermediates, since the cellular constituents are more dilute in the homogenate preparation than in the intact cells. Another possibility is that these results may be indicative of a difference in the cellular and subcellular components present in the homogenates as compared with the dissociated cells.