Characterization of Ca2+ uptake in a subcellular membrane fraction of Herpetomonas sp. promastigotes.
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 microm 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 microm and Km2=29.6+/-6.8 microm. 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.
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJ Brazil.
C L
CL
Sodré
B L M
BL
Moreira
J R
JR
Meyer-Fernandes
P M L
PM
Dutra
A H C S
AH
Lopes
H M
HM
Scofano
H
H
Barrabin
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D008018:Life Cycle Stages / Q000502:physiology*; D010070:Oxalates / Q000494:pharmacology; D013347:Subcellular Fractions; D014351:Trypanosomatina / Q000254:growth & development* / Q000378:metabolism*
D007476:Ionophores; D010070:Oxalates; D002118:Calcium