Penetration of biofilm and targeted, sustained release from liposomes can explain the superior in vivo efficacy of inhaled liposomal amikacin versus free drug observed in a 14 day infection model. Inhaled liposomal amikacin may represent an important therapy for chronic lung infections.

Annexin V is a Ca(2+)-dependent phospholipid-binding protein that may have one or more membrane-related functions, including inhibition of blood coagulation. The fluorescence of the single tryptophan of annexin V was used to monitor Ca2+ and/or phospholipid binding in terms of emission wavelength, emission intensity, and susceptibility to acrylamide quenching. In the absence of phospholipid, Ca2+ titration showed a strong red shift of the wavelength of maximal emission to approximately 345 nm, where a small increase in intensity occurred and was half maximal at approximately 3 mM Ca2+. The Stern-Volmer quenching constant due to acrylamide was only 5.2 M-1 for annexin V alone, indicating limited aqueous exposure of the tryptophan, but 36 M-1 for a Ca(2+)-bound form, indicating full exposure. Binding to both negatively charged and zwitterionic phospholipids was accompanied by a very large increase in fluorescence emission intensity, a red shift, and low exposure to acrylamide. Calculated concentrations of Ca2+ near the surface of negatively charged vesicles suggested that the exposure of tryptophan by Ca2+ binding to annexin V was sufficient for binding of the protein to all vesicles tested, including those composed of oleic acid and phosphatidylcholine (PC), but not to those composed of pure PC. When binding to PC was monitored, the phenomena associated with phospholipid binding were observed separately, at higher Ca2+ concentration, from the red shift and the high exposure to acrylamide due to Ca2+ binding alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The annexins are a novel class of calcium-dependent membrane binding proteins with highly homologous sequences and similar binding characteristics. In order to better define structural parameters of the membrane-bound form, the localization of tryptophan residues in several of these proteins was studied by use of quenchers of their intrinsic fluorescence. Lipocortin I contains a single tryptophan located near its N-terminus, while the single tryptophan in lipocortin V is located in a repeated consensus sequence. Calcium-dependent binding to vesicles composed of 50% egg phosphatidylcholine and 50% bovine brain phosphatidylserine was accompanied by an increase in emission intensity resulting from a relief of internal quenching. The tryptophan fluorescence of bound lipocortin I was nearly unaffected by substituting the quencher 1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphocholine (5-PC) for egg phosphatidylcholine, while that of the lipocortin V tryptophan was quenched significantly. With the quenching doxyl spin-label located deeper in the bilayer at the 12- and 16-positions of the acyl chain, the quenching was progressively weaker, suggesting an interfacial location for the tryptophan of lipocortin V. The same experiments with lipocortin I show almost no quenching in any case, suggesting that this tryptophan near the amino terminus is protected or oriented away from the membrane surface. Data on the bovine liver calelectrins are also presented showing that endonexin also has a tryptophan residue that interacts strongly with phospholipids.

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)

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