The potency of annexin V to transport Ca 2+ ions across phospholipid membranes was investigated, using large unilamellar phospholipid vesicles loaded with the Ca z+ indicator fura-2. It was demonstrated that annexin V leaves the vesicle membranes intact when added in the presence of 1 mM Ca 2+. However, if the vesicles were first incubated with aunexin V in the absence of Ca 2+, subsequent addition of Ca 2+ produced a fluorescence signal due to binding of Ca 2+ to fura-2. Centrifugation of the vesicle suspension immediately thereafter showed that this signal originated from the supernatant and not from the sedimented vesicles. Our results show that annexin V causes loss of vesicle integrity in the absence of Ca 2+, and leakage of trapped fura-2, rather than inward Ca 2+ transport. Bovine serum albumin or Ca 2+ concentrations higher than 2.5 mM also caused such fura-2 leakage. Apparently, calcium-dependent binding of annexin V to the membrane prevents aspecific membrane damage caused by this protein.Key words: Annexin V; Fura-2; Large unilamellar vesicle; Membrane damage; Calcium channel was interpreted as formation of calcium channels promoting influx of calcium into the vesicles. Ultrastructural analysis has revealed a hydrophilic pore in the annexin V molecule that could serve as an ion-selective filter [5,7,8].In view of the earlier mentioned peripheral binding and rapid desorption of annexin V, the formation of transbilayer channels of this protein seems surprising. Also, using black lipid membranes [9,10] of much larger surface area than membranes on micropipettes, we were unable to confirm calcium channel formation by annexin V in the presence of 1 mM calcium (H. Miedema, IMBS, Amsterdam, unpublished results).Here we report the investigation of membrane perturbing properties of annexin V in the LUV system as described by Berendes [6]. The obtained results show that at low calcium concentrations annexin V perturbs membranes such that they become leaky. In contrast, at higher calcium concentrations annexin V promotes membrane integrity.
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