Abstract:
:Annexin V is a Ca(2+)-dependent, phospholipid-binding protein that may have one or more membrane-related functions. The binding of annexin V to phospholipids in a detergent micelle matrix was studied to attempt to determine directly the stoichiometry of specific phospholipid-binding sites and the importance of negative charge. When annexin V binds to phospholipids, a large increase (severalfold) of the emission intensity of tryptophan 187 is observed. This intensity change was used to monitor the binding to phosphatidylcholine (PC) or phosphatidylserine (PS) at varying ratios with the detergent, octaethylene glycol monododecyl ether (C12E8). No binding to PC alone in these micelles could be observed, while approximately 10 PS molecules per micelle were required to observe binding. However, inclusion of negatively charged amphiphiles in the micelles, such as oleic acid or dodecyl sulfate, allowed the observation of binding to PC and decreased the number of phospholipids per micelle necessary for binding to both PS and PC. By including increasing proportions of dodecyl sulfate in the C12E8 micelles, a minimum average number of PS or PC per micelle of approximately 3-4 was required for complete binding. Labeling with photoreactive phospholipids under similar conditions led to an average of approximately 4-5 phospholipids covalently bound per annexin V monomer. Since annexin V has four similar domains, it is reasonable to suggest that one phospholipid binding site is associated with each domain, although as few as three functional domains may be sufficient for binding. Efficient binding required certain structural features of the phospholipid, including a phosphate group, an sn-2 acyl chain, and at least a few carbons on the sn-2 chain. Phospholipid headgroups were almost irrelevant, except for important surface charge effects on the interfacial ionic double layer. A negative surface charge on the micellar aggregate nonspecifically increases the Ca2+ concentration near the micelle surface and may also directly enhance the affinity of annexin V for phospholipids, as shown by the decreased two-dimensional phospholipid concentration necessary for binding. The ability to bind to zwitterionic phospholipids in the presence of a nonspecific negative surface charge may be relevant to the extracellular functions of this protein. Relatively weak individual phospholipid-binding sites that easily exchange were observed, suggesting rapid exchange of phospholipids between the sites on membrane-bound annexin V. These data suggest a working hypothesis that includes approximately four binding sites specific for phospholipid phosphate groups and sn-2 acyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Meers P,Mealy Tdoi
10.1021/bi00094a030subject
Has Abstractpub_date
1993-11-02 00:00:00pages
11711-21issue
43eissn
0006-2960issn
1520-4995journal_volume
32pub_type
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