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. Sever al 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, sodi um tartrate and okadaic acid, (b) inhibition by sodium vanadate, and ( c) activation by spermidine. Additionally, T. brucei PTPase activity p resented two novel features, an acidic pH optimum at pH 4.0-5.0 and a very low K-m value (2.5 nM) for the specific synthetic substrate, Tyr( P)Raytide. Higher K-m values of 170 nM for Tyr(P)-RCML (RCML, reduced, carboxamido-methylated and maleylated lysozyme) and of 3 mM for the n on-specific inorganic substrate p-nitrophenyl phosphate, suggested tha t the PTPase activity of T. brucei was substrate specific. Reconstitut ion experiments on bloodstream-stage membrane proteins revealed that t hree polypeptides of 148, 115 and 72 kDa contained vanadate-inhibitabl e PTPase activity. Modulator assays revealed that the 72-kDa protein w as 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. br ucei was life-cycle-stage regulated. Neither the whole membrane fracti on nor the reconstituted proteins of the procyclic insect stage dephos phorylated tyrosine residues.