The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles. Measurement of vesicle fusion by fluorescence energy transfer

Abstract

1. GTP-promoted fusion between microsomal vesicles was studied by using fluorescence-resonance-energy transfer between the fluorescent membrane probes octadecanoyl-aminofluorescein and octadecylrhodamine. 2. The fluorescence increase after GTP addition does not require the presence of ATP, is unaffected by changes in free [Ca2+] in the range 10 .mu.M-1 nM, but requires Mg2+, although higher Mg2+ concentrations are inhibitory. 3. In terms of requirements for poly(ethylene glycol), dependence on GTP concentration and inhibition by high Mg2+ concentrations, there is excellent correlation between rate of increase in fluorescence and rate of GTP-promoted Ca2+ efflux measured under Ca2+ transport conditions. 4. The observations support our previous conclusions that GTP-induced membrane fusion plays a major role in causing GTP-promoted Ca2+ efflux from microsomal vesicles.