ANNEXIN-II BINDS TO THE MEMBRANE OF A549-CELLS IN A CALCIUM-DEPENDENTAND CALCIUM-INDEPENDENT MANNER

We investigated the nature of annexin II binding ro the biological mem branes using a lung epithelium-derived cell line A549. The cytosolic a nd membrane fractions of A549 cells were separated in the presence of 5 mM EGTA. Both fractions contain annexin II monomer and tetramer as e valuated by western blots using specific monoclonal antibodies against p36 and p11 subunits of annexin II. A substantial amount of annexin I I was associated with the membrane fraction even after extensive washi ng with EGTA buffer, indicating the presence of two pools of annexin I I. The EGTA-resistant membrane-bound annexin II could be partially ext racted by 1% Triton X-100 or 60 mM n-octyl-beta-D-glucopyranoside, and completely by 30 mM CHAPS or 0.1% deoxycholate. This fraction of anne xin II was also extracted by 0.1 M Na2CO3, pH 11 and partitioned into the aqueous phase after being treated with Triton X-114, demonstrating that the EGTA-resistant annexin II is a peripheral membrane protein. When the cells were lysed in varying concentrations of Ca2+, annexin I I translocated from cytosolic fraction to membrane fraction at 4-25 mu M Ca2+. To identify proteins closely associated with annexin II the m embrane fraction was treated with the bifunctional chemical cross-link er disulfosuccinimidyl tartarate, followed by western blot analysis us ing anti-p36 or anti-p11 antibodies. We find that both p36 and p11 wer e cross-linked to a 51 kDa protein. In addition, p11 also binds to sev eral proteins with molecular mass of 91,65, 40 and 36 kDa. Our results suggest that annexin II may bind to the A549 cell membranes via speci fic membrane-associated proteins. (C) 1997 Elsevier Science Inc.