Localization of Glycerol‐3‐Phosphate Oxidase in the Mitochondrion and Particulate NAD⁺‐Linked Glycerol‐3‐Phosphate Dehydrogenase in the Microbodies of the Bloodstream Form of <i>Trypanosoma brucei</i>

Abstract

We have studied the intracellular location of glycerol‐3‐phosphate oxidase and NAD + ‐linked glycerol‐3‐phosphate dehydrogenase, two enzymes involved in the very active dihydroxyacetone phosphate:glycerol‐3‐phosphate cycle in Trypanosoma brucei . Isopycnic centrifugation of a large‐granule fraction resulted in a 10–20‐fold purification of the oxidase and showed that the enzyme behaved like the mitochondrial ATPase, isocitrate dehydrogenase and the particulate malate dehydrogenase. The activity profiles of these four enzymes in sucrose gradients were identical with maximal activity at a density of 1.17 g/cm 3 . Electron micrographs of a fraction equilibrating at this density only showed mitochondrial profiles. The particle‐bound NAD + ‐linked glycerol‐3‐phosphate dehydrogenase activity was found to band at a density of 1.23 g/cm 3 in sucrose. A fraction equilibrating at this density was highly enriched in microbodies; it contained few mitochondrial profiles and less than 3% of the specific oxidase activity of the mitochondrial fraction. Because of the absence of microbody markers in T. brucei we turned to the insect trypanosome Crithidia luciliae , which contains a high activity of the microbody marker catalase. In C. luciliae part of the catalase activity bands at the same density as the particle‐bound NAD + ‐linked glycerol‐3‐phosphate dehydrogenase (Q = 1.26 g/cm 3 ). From these results we conclude that in T. brucei bloodstream forms glycerol‐3‐phosphate oxidase is located in the mitochondrion and not in the microbodies, as proposed by a number of other investigators, and that the particle‐bound NAD + ‐linked glycerol‐3‐phosphate dehydrogenase is located within the microbodies. It is likely that this highly active dehydrogenase plays an essential role in glycolysis.