One can estimate an appar ent dissociation constant for the protein binding step at a constant calcium concentration from Equation 5. To estimate how large the effect of transmembrane potential might be under selleck screening library physiologic conditions, we estimated a Kd, app that would pertain to protein membrane binding interactions occurring at a constant free calcium concen tration of 1. Inhibitors,Modulators,Libraries 25 mM. The theoretical calculation indicates that the apparent Kd will increase by about a factor of 10. How much this will alter annexin V binding depends in turn on the Inhibitors,Modulators,Libraries apparent affinity for annexin V, and the concentration of annexin V used in the assay. The affinity of annexin V for cells is much less than for the phospholipid vesicles with 25% PS used in Table 1, with pKd values around 30 and apparent Kd values reported in the range from about 5 to 30 nM.
Figure 1 shows a family of theoretical curves for the situa tion where the Kd, app becomes tenfold tighter as a result of changes in the membrane potential. When the concentra tion of annexin V is far below the apparent Kd, the effect of membrane Inhibitors,Modulators,Libraries potential can be very large, but as the concentration of annexin V increases above the apparent Kd, the relative buffer increases the average annexin V binding of the annexin positive popu lation, but does not alter the percentage of annexin nega tive cells in either untreated or UV treated cells. These results are as predicted from the vesicle binding studies above, i. e. making transmembrane potential less negative would increase the binding of annexin V to the external face Inhibitors,Modulators,Libraries of the plasma membrane.
The magnitude of the change in binding is also reasonably consistent with the theoretical predictions of Figure 1 at an annexin V con centration of 30 nM and a starting Kd, app of 10 nM, Figure 1 predicts an increase in binding of about 1. 3 fold, con sistent with the observed value of about 1. 4. depolarizationcalculationaffinityincreaseincreasedconcentrationaffinity Inhibitors,Modulators,Libraries due increase in binding becomes smaller and smaller. Although this theoretical analysis does not take account of all the complexities of binding to natural cell membranes, it does provide a general guide to the magnitude of the effect that might be observed with living cells under phys iologic conditions. Depolarization increases binding of annexin V to cells undergoing apoptosis We next tested whether living cells would show altered annexin V binding as a function of changes in membrane potential.
Jurkat thorough T leukemia cells provide a good model system, as they normally have a significant resting mem brane potential of about 60 mV and also expose PS early in apoptosis. When Jurkat cells are treated with UV light, they start to undergo apoptosis, as shown by the development of a population of annexin positive cells.