Effects of divalent ions on vesicle-vesicle fusion studied by a new luminescence assay for fusion

Abstract

A new assay has been developed for vesicle-vesicle fusion based upon the mixing of intravesicular contents of two sets of vesicles. Purified firefly luciferase and MgCl 2 were incorporated into one set of vesicles (LV) and ATP into the other (AV). Vesicles were prepared from soybean phospholipids. The luminescence that resulted from hydrolysis of ATP by luciferase was measured to determine the extent of mixing of the intravesicular contents. In the absence of divalent ions, incubation of a mixture of LV and AV did not produce luminescence. However, if Ca ++ or other divalent ions were present at millimolar concentrations, luminescence occurred. The luminescence did not result from extravesicular reaction of vesicle contents that had leaked into the medium. Instead, luminescence resulted from the mixing of intravesicular spaces of AV and LV in fused vesicles. Optical density changes and negative stain electron microscopy indicated that Ca ++ induced extensive aggregation of vesicles. However, quantitation of the maximum possible luminescence indicates that only a small percentage (less than 1%) of the vesicles actually fused in a fusion experiment.