Characterization of a life-cycle-stage-regulated membrane protein tyrosine phosphatase in Trypanosoma brucei.
We report the first characterization of plasma-membrane-bound tyrosine phosphatase activity in the haemoprotozoan. Trypanosoma brucei. Several enzymic properties of the membrane fraction were identical to other protein tyrosine phosphatases (PTPases), such as (a) insensitivity to inhibitors of other protein phosphatases, including tetramisole, sodium tartrate and okadaic acid, (b) inhibition by sodium vanadate, and (c) activation by spermidine. Additionally, T. brucei PTPase activity presented two novel features, an acidic pH optimum at pH 4.0-5.0 and a very low Km value (2.5 nM) for the specific synthetic substrate, Tyr(P)Raytide. Higher Km values of 170 nM for Tyr(P)-RCML (RCML, reduced, carboxamidomethylated and maleylated lysozyme) and of 3 mM for the non-specific inorganic substrate p-nitrophenyl phosphate, suggested that the PTPase activity of T. brucei was substrate specific. Reconstitution experiments on bloodstream-stage membrane proteins revealed that three polypeptides of 148, 115 and 72 kDa contained vanadate-inhibitable PTPase activity. Modulator assays revealed that the 72-kDa protein was responsible for the observed spermidine stimulation, but indicated that the modulator profile of the 148-kDa protein was most similar to the whole membrane fraction. Furthermore, the PTPase activity of T. brucei was life-cycle-stage regulated. Neither the whole membrane fraction nor the reconstituted proteins of the procyclic insect stage dephosphorylated tyrosine residues.
Bakalara
N
N
Laboratorie d'Immunologie et Parasitologie Moléculaire, Université Bordeaux II, France.
Seyfang
A
A
Davis
C
C
Baltz
T
T
D000818:Animals; D002453:Cell Cycle; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011489:Protein Denaturation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013095:Spermidine / Q000494:pharmacology; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010455:Peptides; C071998:Raytide; C008644:nitrophenylphosphate; D017027:Protein Tyrosine Phosphatases; D013095:Spermidine