Identification and partial purification of a stage-specific 33 kDa mitochondrial protein as the alternative oxidase of the Trypanosoma brucei brucei bloodstream trypomastigotes.
The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.
Chaudhuri
M
M
Division of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208, USA.
Ajayi
W
W
Temple
S
S
Hill
G C
GC
D000818:Animals; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008970:Molecular Weight; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins; D010944:Plants / Q000201:enzymology / Q000276:immunology; D011190:Potassium Cyanide / Q000494:pharmacology; D012457:Salicylamides / Q000494:pharmacology; D014344:Trypanocidal Agents / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D000099188:Alternative Oxidase
D000911:Antibodies, Monoclonal; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011190:Potassium Cyanide; D012457:Salicylamides; D014344:Trypanocidal Agents; D000099188:Alternative Oxidase; C005703:salicylhydroxamic acid