The calcium-dependent interaction of several annexins with membranes was studied. A novel technique was developed that allowed estimation of calcium binding to aggregating systems. This consisted of immobilized phospholipids on phenyl-Sepharose. Proteins associated with this affinity gel in the presence of radioactive calcium and were eluted with the same buffer containing excess EGTA. This produced an elution profile with a peak of excess calcium. Protein extinction coefficients were estimated in order to quantitate the protein more accurately. Association of annexin II with the membrane was of very high affinity and involved a calcium stoichiometry of 11 +/- 1 at 12.5 mu M free Ca2+ and 10 +/- 2 at 50 mu M free Ca2+. (AII)(2)(p11)(2), a heterotetramer of two annexin II and two p11 subunits, bound 12 +/- 1 Ca2+ at 12.5 mu M Ca2+ and 15 +/- 1 Ca2+ at 50 mu M Ca2+. These stoichiometries showed a pattern of ''all or none'' calcium binding where the number of calcium ions bound to a protein-membrane complex was virtually independent of the free calcium concentration or the density of protein on the membrane. (AII)(2)(p11)(2) contains two annexin II subunits so that calcium stoichiometry was not directly related to the number of potential sites. (AII)(2)(p11)(2) required less calcium to support membrane binding than did annexin II, Thus, dimerization of the membrane binding unit may be needed for annexins to function at intracellular calcium levels. Annexin VI contains twice as many putative calcium binding units as annexin V and the same pattern of behavior occurred for this pair of proteins. At 25 mu M free calcium, (AII)(2)(p11)(2) alone bound no detectable calcium (<0.1 mol of calcium/mol of protein) and annexin II bound only 0.3-0.6 calcium ions. These results suggested a general pattern of behavior for annexin proteins where each component, protein and membrane, provides a part of the many calcium binding sites. Partial calcium binding sites on the individual components may not correlate precisely with calcium binding sites of the complex.
