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.
Sodré
C L
CL
Instituto de Bioquímica Médica, ICB/CCS, UFRJ, RJ Brazil.
Moreira
B L M
BL
Meyer-Fernandes
J R
JR
Dutra
P M L
PM
Lopes
A H C S
AH
Scofano
H M
HM
Barrabin
H
H
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