Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei |
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Authors: | Cedro Fernandes Eloise Mauro Granjeiro José Mikio Taga Eulázio Meyer-Fernandes José Roberto Aoyama Hiroshi |
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Affiliation: | Departamento de Bioquímica, Universidade Federal de S?o Paulo, 04023900 S?o Paulo, SP, Brazil. eloise@cec.iac.br |
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Abstract: | Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [(32)P]phosphocasein, is inhibited weakly by NaF and tartrate but strongly by vanadate. In this work, we describe the presence of an external phosphatase activity in intact bloodstream forms of T. brucei. With p-nitrophenyl phosphate (pNPP) as substrate, these intact cells produced 3-5 nmol pNP min(-1) mg(-1), linearly for up to at least 30 min. The activity was not significantly increased by Mg(2+), Mn(2+), Ca(2+) and Co(2+), but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn(2+) and was insensitive to okadaic acid. Membrane-enriched fractions of parasites contained an acid phosphatase activity, with a pH optimum in the range of 4.5-5.5. This activity hydrolyzed phosphotyrosine (40 nmol phosphate min(-1) mg(-1)) better than phosphothreonine or phosphoserine. Partial purification of this phosphatase yielded a single activity band following gel electrophoresis, a K(m) value of 0.29 mM with pNPP and was insensitive to the Fe(2+)/H(2)O(2)/ascorbate system. |
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Keywords: | Phosphotyrosine phosphatase Bloodstream forms Trypanosoma brucei |
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