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

Annexins VI and V are members of the annexin family of proteins that b ind to phospholipid membranes in a calcium-dependent manner. The dynam ics of protein, calcium, and phospholipid assembly and dissociation we re investigated by stopped-flow. At relatively low calcium levels, the kinetics of the binding reaction were sensitive to calcium concentrat ion. However, in the presence of saturating levels of calcium and at r elatively low protein/vesicle (w/w) ratios (0.4 or lower), the binding reactions were rapid and the rate constants were comparable to the co llisional 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 s mall unilamellar vesicles (about 31 nm in diameter). These constants a re expressed on the basis of vesicle concentration. These limiting ass ociation rate constants were not sensitive to the phosphiolipid compos ition of the vesicles. In contrast, at these calcium levels, protein d issociation was so slow that the complexes could be regarded as stable . However, individual calcium ions that were bound to the complexes ap peared to exchange rapidly with ions in bulk solution. EGTA-induced pr otein dissociation was rapid with first-order rate constants ranging f rom 10 to 50 s(-1) These were dependent on the membrane composition an d on the protein type (annexin VI or V). Variations in this dissociati on process were found to complement the calcium concentration needed t o support annexin-membrane association; increasing the acidic phosphol ipid component or partially replacing phosphatidylcholine by phosphati dylethanolamine in the membrane decreased both the EGTA-induced dissoc iation rate and the calcium concentration needed to support binding. T his correlation suggested that the rate of EGTA-induced protein dissoc iation was actually a function of the rate of calcium dissociation fro m the protein-calcium-phospholipid complexes. These behaviors were con sistent with a facile interaction between the protein and membrane thr ough a large number of calcium ions.