Glucose catabolism in African trypanosomes. Evidence that the terminal step is catalyzed by a pyruvate transporter capable of facilitating uptake of toxic analogs.
The protozoan parasite Trypanosoma brucei derives its metabolic energy exclusively from a unique type of glycolysis in which pyruvate derived from glucose catabolism is released into the host bloodstream. In this study, this terminal metabolic step has been examined in detail. Pyruvate release from trypanosomal cells supplied with glucose is very rapid, proceeding with an apparent Vmax of 214 nmol x min-1 x mg-1. Counterflow experiments with [14C]pyruvate demonstrate that this metabolic end product can be taken up by actively metabolizing cells consistent with the presence of a plasma membrane transporter. The findings that [14C] acetate exhibits a much lower capacity for cell entry and that the structural analog alpha-cyano-3-hydroxycinnamic acid inhibits pyruvate release provide additional support for the presence of a pyruvate transporter. The substrate analog and alkylating agent 3-bromopyruvate inhibits completely both cell motility and pyruvate release. Surprisingly, however, it is a poor inhibitor of pyruvate transport per se. Rather, its preferential site of action and that of iodoacetic acid were identified by radiolabeling studies and microsequence analysis as glyceraldehyde-3-phosphate dehydrogenase. In extending these studies, 3-bromopyruvate was found to be over 20 times less effective in inhibiting glyceraldehyde-3-phosphate dehydrogenase in intact erythrocytes than in trypanosomal cells. However, in sonicated preparations from both cell types, the enzyme exhibits nearly identical sensitivities to inhibition by 3-bromopyruvate. Experiments reported here provide the first direct evidence that pyruvate release in African trypanosomes is catalyzed by a specific transport system and implicate this transporter as a vehicle for delivering toxic alkylating agents into trypanosomal cells.
Barnard
J P
JP
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Reynafarje
B
B
Pedersen
P L
PL
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D005947:Glucose / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D027501:Monocarboxylic Acid Transporters; D010042:Ouabain / Q000494:pharmacology; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000302:isolation & purification / Q000378:metabolism*
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D027501:Monocarboxylic Acid Transporters; D015800:Protozoan Proteins; D011773:Pyruvates; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010042:Ouabain; C017092:bromopyruvate; D008985:Monensin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005947:Glucose; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid