title: Absence of substrate channeling in the glycosome of Trypanosoma brucei.
abstract: Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966-6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of alpha-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of alpha-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5-5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2-deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. All results suggest an absence of substrate channelings among the glycolytic enzymes in the glycosome of T. b. brucei bloodstream form.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003847:Deoxyglucose / Q000378:metabolism; D004120:Dimethyl Suberimidate; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D006019:Glycolysis; D008830:Microbodies / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000648:ultrastructure
chemical_list: D003432:Cross-Linking Reagents; D005635:Fructosediphosphates; D005994:Glycerophosphates; D009097:Multienzyme Complexes; D009243:NAD; D004120:Dimethyl Suberimidate; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; C029620:alpha-glycerophosphoric acid; D006597:Fructose-Bisphosphatase; D005947:Glucose; C029063:fructose-1,6-diphosphate