Monoclonal antibodies specific to a Ca2+-bound form of lipocortin I distinguish its Ca2+-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2

摘要

pLipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects iin vitro/i. Its actual role iin vivo/i, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both iin vivo/i and iin vitro/i. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to iEscherichia coli/i membranes and to phosphatidylserine iin vitro/i. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I iin vivo/i./p