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Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes

Published online by Cambridge University Press:  16 April 2009

C. L. SODRÉ ,
B. L. M. MOREIRA ,
J. R. MEYER-FERNANDES ,
P. M. L. DUTRA ,
A. H. C. S. LOPES ,
H. M. SCOFANO  and
H. BARRABIN
C. L. SODRÉ
Affiliation:
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJBrazil Instituto Oswaldo Cruz, FIOCRUZ, RJ, Brazil
B. L. M. MOREIRA
Affiliation:
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJBrazil
J. R. MEYER-FERNANDES
Affiliation:
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJBrazil
P. M. L. DUTRA
Affiliation:
Disciplina de Parasitologia, DMIP, FCM, UERJ, RJ, Brazil
A. H. C. S. LOPES
Affiliation:
Departamento de Microbiologia Geral, Instituto de Microbiologia, CCS, UFRJ, RJ, Brazil
H. M. SCOFANO
Affiliation:
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJBrazil
H. BARRABIN*
Affiliation:
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJBrazil
*
*Corresponding author: Departamento de Bioquímica Médica, ICB/CCS, Cidade Universitária, Universidade Federal do Rio de Janeiro, CEP 21941-590 Rio de Janeiro, RJBrasil. E-mail: [email protected]
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Summary

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0·5 of 0·1 μm and a Hill number (nH)=2·8±0·4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7·0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0·25±0·19 μm and Km2=29·6±6·8 μm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

Keywords

Herpetomonastrypanosomatidscalciumendoplasmic reticulumSERCAATP dependencevanadateoxalateCa2+ uptake
Type
Research Article
Information
Parasitology , Volume 136 , Issue 6 , May 2009 , pp. 657 - 663
Copyright
Copyright © Cambridge University Press 2009

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