Mouse ornithine decarboxylase is stable in Trypanosoma brucei.
The cDNA encoding mouse ornithine decarboxylase (ODC) was incorporated into a transforming vector pTSA-NEO2 carrying a procyclic acidic repetitive protein promoter and a neomycin phosphotransferase gene. The plasmid thus constructed, pMOD300, was introduced into the procyclic forms of Trypanosoma brucei via electroporation, and the transformants, selected under G418, expressed an ODC activity 100 times above the background level. Contrary to the commonly observed short half-life of mouse ODC in mammalian cells, however, the mouse ODC activity expressed in T. brucei remained stable for at least 6 h when protein synthesis was inhibited by cycloheximide. Pulse labelings and chase experiments with the irreversible ODC inhibitor [3,4-3H]difluoromethylornithine followed by gel electrophoresis, or with L-[35S] methionine followed by immunoprecipitation and gel electrophoresis indicated that the stable mouse ODC expressed in T. brucei has the same subunit molecular weight as the native enzyme. By an in vitro assay of protein stability in rabbit reticulocyte lysates (Loetscher, P., Pratt, G., and Rechsteiner, M. (1991) J. Biol. Chem. 266, 11213-11220), the native mouse ODC and the enzyme expressed in T. brucei had the same degree of instability. Thus, the mouse ODC expressed in T. brucei is probably identical to the native mouse ODC. Its remarkable stability in T. brucei must be due to the absence in trypanosomes of the proteolytic machinery present in mammalian cells responsible for rapid degradation of mouse ODC.
Bass
K E
KE
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Sommer
J M
JM
Cheng
Q L
QL
Wang
C C
CC
D000818:Animals; D004247:DNA / Q000235:genetics; D000518:Eflornithine / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D004795:Enzyme Stability; D051379:Mice; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D010957:Plasmids; D011233:Precipitin Tests; D014170:Transformation, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D065108:Ornithine Decarboxylase Inhibitors; D004247:DNA; D009955:Ornithine Decarboxylase; D000518:Eflornithine