title: The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei.
abstract: Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3-phosphoglycerate. The multienzyme reaction which produces 2 alpha-glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha-glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions.
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000818:Animals; D050260:Carbohydrate Metabolism; D002451:Cell Compartmentation; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D005991:Glycerol Kinase / Q000378:metabolism*; D005994:Glycerophosphates / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010732:Phosphofructokinase-1 / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D005994:Glycerophosphates; D009243:NAD; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C029620:alpha-glycerophosphoric acid; D005954:Glucosephosphate Dehydrogenase; D010770:Phosphotransferases; D006593:Hexokinase; D010732:Phosphofructokinase-1; D005991:Glycerol Kinase; D005947:Glucose