Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei

Abstract

Procyclic forms of Trypanosoma brucei possess a phosphatase activity on their external cell surface. This activity, while it dephosphorylates [P-32]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 Mg2+, Mn2+, Ca2+ and Co2+, but was inhibited by vanadate, NaF, p-chloromercuribenzoate and Zn2+ 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 Fe2+/H2O2/ascorbate system. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.