calcimycin and dioleoylphosphatidic-acid

Calcium binds to dioleoylphosphatidate/dioleoylphosphatidylcholine (DOPA/DOPC) (20:80, mol%) multilamellar vesicles in the presence of a calcium ionophore with stoichiometry of about 0.6 nmol calcium per nmol phosphatidate and an apparent dissociation constant of about 1.7 mM. Experiments on the behaviour of monomolecular films at an air/water interface show that calcium-phosphatidate binding results in a decrease in the area of the polar region of the phosphatidate molecule, probably caused by headgroup dehydration and partial charge neutralization. At calcium concentration higher than about 3 mM calcium neutralizes the negatively charged membrane surface of DOPA/DOPC (20:80, mol%) large unilamellar vesicles, and vesicle aggregation is observed. At 10 mM of calcium this results in a low level of vesicle fusion. These observed processes are not attended with calcium-induced phosphatidylcholine transbilayer movement in the membranes of DOPA/DOPC (20:80, mol%) large unilamellar vesicles. When these findings are compared with the results of a previous study on the permeability behaviour of large unilamellar vesicles of the same phospholipid composition under comparable conditions (Smaal, E.B., Mandersloot, J.G., De Kruijff, B. and De Gier, J. (1986) Biochim. Biophys. Acta 860, 99-108) the following conclusions can be drawn. At low millimolar calcium concentrations (less than 2.5 mM) calcium does not occupy all the binding sites of the membrane, no membrane-membrane interactions are observed and a selective translocation of calcium and calcium-chelating anions is appearing. The mechanism of this translocation may be explained by the formation of uncharged dehydrated complexes of calcium, phosphatidate and calcium chelator, which can pass the membrane via transient occurring non-bilayer structures. Between 3 and 10 mM of calcium an a selective permeability increase of the vesicular membrane is found, which is not a consequence of vesicle fusion but apparently of vesicle aggregation, possibly causing packing defects in the membrane.

Topics: Calcimycin; Calcium; Freeze Fracturing; Membrane Fusion; Membrane Lipids; Membranes, Artificial; Permeability; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylglycerols

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.

Topics: Arsenazo III; Azo Compounds; Biological Transport, Active; Calcimycin; Calcium; Lipid Bilayers; Liposomes; Methods; Methoxyhydroxyphenylglycol; Permeability; Phosphatidic Acids; Phosphatidylcholines; Phospholipids

The ionophoretic capabilities of dioleoylphosphatidic acid (DOPA) for transporting calcium across phospholipid bilayers have been investigated. Calcium uptake by large unilamellar vesicles is shown to depend on the presence of DOPA. This uptake is sensitive to the nature and concentration of calcium chelators in the vesicle interior, indicating that accumulation results from DOPA-mediated translocation of calcium across the membrane. Further, it is shown that characteristics of DOPA-mediated Ca2+ uptake are similar to those observed for the fungal calcium ionophore, A23187.

Topics: Biological Transport; Calcimycin; Calcium; Egtazic Acid; Ionophores; Kinetics; Liposomes; Phosphatidic Acids