Annexin II binds to the membrane of A549 cells in a calcium-dependent and calcium-independent manner.
We investigated the nature of annexin II binding to the biological membranes using a lung epithelium-derived cell line A549. The cytosolic and membrane fractions of A549 cells were separated in the presence of 5 mM EGTA. Both fractions contain annexin II monomer and tetramer as evaluated by western blots using specific monoclonal antibodies against p36 and p11 subunits of annexin II. A substantial amount of annexin II was associated with the membrane fraction even after extensive washing with EGTA buffer, indicating the presence of two pools of annexin II. The EGTA-resistant membrane-bound annexin II could be partially extracted 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 annexin 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 II translocated from cytosolic fraction to membrane fraction at 4-25 microM Ca2+. To identify proteins closely associated with annexin II the membrane fraction was treated with the bifunctional chemical cross-linker disulfosuccinimidyl tartarate, followed by western blot analysis using anti-p36 or anti-p11 antibodies. We find that both p36 and p11 were cross-linked to a 51 kDa protein. In addition, p11 also binds to several 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 specific membrane-associated proteins.
Liu
L
L
Institute for Environmental Medicine, University of Pennsylvania, Medical Center, Philadelphia 19104, USA.
Tao
J Q
JQ
Zimmerman
U J
UJ
D017306:Annexin A2 / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002254:Carbonates / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D013388:Succinimides / Q000494:pharmacology
D017306:Annexin A2; D002254:Carbonates; D003432:Cross-Linking Reagents; D003902:Detergents; D013388:Succinimides; C072854:bis(sulfosuccinimidyl)tartrate; C005686:sodium carbonate; D004533:Egtazic Acid; D002118:Calcium