Annexin IV reduces the rate of lateral lipid diffusion and changes the fluid phase structure of the lipid bilayer when it binds to negatively charged membranes in the presence of calcium.
Bovine annexin IV (endonexin) was bound to supported planar bilayers composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) in the first monolayer facing the substrate, and varying mole fractions of POPC, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and small amounts of the fluorescent lipid analogs NBD-PC or NBD-PG in the second monolayer facing the large aqueous compartment. Lateral diffusion coefficients and mobile fractions of these phospholipids were measured by fluorescence recovery after photobleaching (FRAP) as a function of protein concentration and lipid composition in the presence of 2 mM CaCl2 or 1 mM EDTA. In the absence of annexin IV, the lateral diffusion coefficients depended only little on the POPC:POPG ratios and were approximately 3.0 microns2/s for NBD-PG (no Ca2+), 2.5 microns2/s for NBD-PG (2 mM Ca2+), and 1.6 microns2/s for NBD-PC (with or without 2 mM Ca2+). In the presence of 2 mM Ca2+ these diffusion coefficients decreased as a function of the added annexin concentration. A transition from a state with "rapid" lipid diffusion to a state with "slow" lipid diffusion occurred at about 80 nM annexin IV and was independent of the POPC:POPG ratio. In addition to reducing the lipid lateral diffusion coefficients, annexin IV also gave rise to two-component lateral diffusion of the lipids in these mixed bilayers. The split of the single diffusion coefficient of NBD-PG into two components occurred at most POPC:POPG ratios upon binding of annexin IV, but required higher annexin concentrations at mole fractions of POPC between 66 and 82 mol % than at high mole fractions of POPG or 90 mol % POPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville 22908.
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R
Gilmanshin
C E
CE
Creutz
L K
LK
Tamm
D000818:Animals; D017314:Annexin A4 / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D004492:Edetic Acid / Q000494:pharmacology; D004563:Electrochemistry; D005456:Fluorescent Dyes; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010777:Photochemistry; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D017314:Annexin A4; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011994:Recombinant Proteins; C060037:1-palmitoyl-2-oleoylglycero-3-phosphoglycerol; D004492:Edetic Acid; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine