Annexin VI, a novel ATP-dependent, phospholipid-binding protein

The determination of surface pressure (π) of a phosphatidylserine (PS) monolayer is used to study the interactions between specific phospholipid classes and various proteins. In the present study we show that ATP, but not ADP, in milimolar concentration ranges stimulate the increase of Δπ in a PS monolayer evoked by annexin VI (AnxVI)/Ca2+ at a moderate initial π (~11 mN/m). The obtained results are consistent with ATP being a functional ligand for AnxVI. To further study the ATP binding site of AnxVI, we have used fluorescein 5’-isothiocyanate (FITC). This is useful in the characterization of nucleotide-binding sites of many membrane integral and cytosolic proteins. Under our experimental conditions FITC did not affect the binding of AnxVI to membranes but abolished the interaction of the protein with ATP insolubilized on agarose. This observation can be interpreted in terms of AnxVI possessing an ATP-binding site functionally similar to nucleotide-binding domains characterized in other ATP-dependent proteins. We also provide evidence that two AnxVI isoforms are expressed constitutively in porcine liver differ from each other in respect to their ATP binding properties.