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 Trypanosoma brucei

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 largegranule 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/cm3. 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/cm3 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 (@ = 1.26 g/cm3).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.

Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.

“…The histogram shows no predominant length classes and all molecules present could represent the degradation products of nuclear DNA or kinetoplast DNA. Several methods have been described for the purification of glycosomes from Trypanosoma brucei (4,21,40). One utilized isopycnic centrifugation of a large-particle preparation in a linear sucrose gradient, where glycosomes equilibrate at a density of 1 .23 g/cm' .…”

“…Since the discovery of these organelles in Trypanosoma brucei (1), they have been found in all major representatives of this family (i.e., T. cruzi (2), Leishmania mexicana (3) and several Crithidia spp. (2,(4)(5)(6)). The glycosomes in T. brucei contain at least nine enzymes involved in or related to glycolysis, which can account for the conversion of glucose to 3-phosphoglycerate plus glycerol.…”

The effect of citrate/cis‐aconitate on oxidative metabolism during transformation of Trypanosoma brucei

“…In bloodstream forms glucose is degraded solely by glycolysis to form two mol pyruvate/mol glucose. Reducing equivalents are transferred to oxygen via a unique mitochondrial glycerol-3-phosphate oxidase [17,18]. The promitochondrion lacks a functional citric acid cycle as well as a cytochrome-linked respiratory chain.…”

An approach to the development of new drugs for African trypanosomiasis.

“…Glucose is metabolized only as far as pyruvate (20); the NAD reduced during glycolysis is regenerated in microbodies by the reduction of dihydroxyacetone phosphate (19,20). The aGP formed is in turn recycled by an oxidase complex which is associated with a rudimentary mitochondria (21). Most of the electrons from the oxidation of aGP are incorporated into water.…”

“…The mitochondrial oxidase is a complex of at least two components, an aGP dehydrogenase and a terminal oxidase (21). The activity of the dehydrogenase can be measured using artificial electron acceptors such as DCIP, and it is not inhibited by either Triton X-100 or benzhydroxamic acids such as SHAM.…”