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

Abstract

SUMMARYATP-dependent Ca2+uptake was studied in a subcellular fraction fromHerpetomonassp. prepared by mechanical disruption and using45Ca2+as a tracer. The uptake was stimulated by Ca2+with a K0·5of 0·1 μmand 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 μmand 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 ofHerpetomonassp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.