Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei.
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.
Cedro Fernandes
Eloise
E
Departamento de Bioquímica, Universidade Federal de São Paulo, 04023900 São Paulo, SP, Brazil. eloise@cec.iac.br
Mauro Granjeiro
José
J
Mikio Taga
Eulázio
E
Meyer-Fernandes
José Roberto
JR
Aoyama
Hiroshi
H
D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008018:Life Cycle Stages; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D018384:Oxidative Stress; D018512:Parasitemia / Q000469:parasitology; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development
D002413:Cations, Divalent; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D019000:Phosphotyrosine; C008644:nitrophenylphosphate; D017027:Protein Tyrosine Phosphatases