KINETICS OF ANNEXIN-VI, CALCIUM, AND PHOSPHOLIPID ASSOCIATION AND DISSOCIATION

Annexins VI and V are members of the annexin family of proteins that bind to phospholipid membranes in a calcium-dependent manner. The dynamics of protein, calcium, and phospholipid assembly and dissociation were investigated by stopped-flow. At relatively low calcium levels, the kinetics of the binding reaction were sensitive to calcium concentration. However, in the presence of saturating levels of calcium and at relatively low protein/vesicle (w/w) ratios (0.4 or lower), the binding reactions were rapid and the rate constants were comparable to the collisional limit, about 1.4 x 10(10) M(-1) s(-1) for large unilamellar vesicles (about 120 nm diameter) and about 2.7 x 10(9) M(-1) S-1 for small unilamellar vesicles (about 31 nm in diameter). These constants are expressed on the basis of vesicle concentration. These limiting association rate constants were not sensitive to the phosphiolipid composition of the vesicles. In contrast, at these calcium levels, protein dissociation was so slow that the complexes could be regarded as stable. However, individual calcium ions that were bound to the complexes appeared to exchange rapidly with ions in bulk solution. EGTA-induced protein dissociation was rapid with first-order rate constants ranging from 10 to 50 s(-1) These were dependent on the membrane composition and on the protein type (annexin VI or V). Variations in this dissociation process were found to complement the calcium concentration needed to support annexin-membrane association; increasing the acidic phospholipid component or partially replacing phosphatidylcholine by phosphatidylethanolamine in the membrane decreased both the EGTA-induced dissociation rate and the calcium concentration needed to support binding. This correlation suggested that the rate of EGTA-induced protein dissociation was actually a function of the rate of calcium dissociation from the protein-calcium-phospholipid complexes. These behaviors were consistent with a facile interaction between the protein and membrane through a large number of calcium ions.