Köhler G, Hering U, Zschörnig O, Arnold K
Institute of Medical Physics and Biophysics, University of Leipzig, Germany.
Biochemistry. 1997 Jul 1;36(26):8189-94. doi: 10.1021/bi9703960.
Annexin V belongs to a class of Ca2+-binding proteins for which different functions in the cell are discussed, e.g., involvement in exocytosis, inhibition of protein kinase C, and calcium channel activity in cartilage matrix vesicles. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study, the interaction of annexin V with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) at low pH was compared to that at neutral pH. Annexin V strongly binds to PS LUV at low pH, whereas at neutral pH 20-100 microM Ca2+ are required to induce binding. This is caused by the different electric charge of the protein. The binding ability of the PS LUV for annexin V is higher at low pH. Binding of annexin V induces dehydration of the vesicle surface and a decrease of the lateral diffusion within the bilayer. While this dehydration is due to vesicle contact at pH 4, at pH 7.4 it is due to surface covering by annexin V. Annexin V promotes the phospholipid intermixing between LUVs at pH 5 and below but inhibits it at pH 7.4. A substitution of up to 80% of the PS by the uncharged phosphatidylcholine does not impair the extent of phospholipid intermixing at pH 4. The high binding capacity of PS LUV, the disappearance of the inhibiting action, and a calculated increase of the annexin V hydrophobicity make it likely that annexin V is able to penetrate into the membrane at low pH. At neutral pH, annexin V molecules act as steric barriers, preventing close apposition of two vesicles. At pH 5, annexin V lowers the threshold concentration of the Ca2+-induced phospholipid intermixing. Such a promotion is well-known for annexin VII (synexin). The effect may be related to the isoelectric points of the two annexins which have been reported as 4.8 (annexin V) and 7.0 (annexin VII), respectively.
膜联蛋白V属于一类钙结合蛋白，人们对其在细胞中的不同功能进行了讨论，例如参与胞吐作用、抑制蛋白激酶C以及软骨基质小泡中的钙通道活性。所有这些功能都与膜联蛋白结合酸性磷脂的能力有关。在本研究中，将膜联蛋白V在低pH值下与由磷脂酰丝氨酸（PS）制备的大单层囊泡（LUV）的相互作用与在中性pH值下的相互作用进行了比较。膜联蛋白V在低pH值下能强烈结合PS LUV，而在中性pH值下则需要20 - 100微摩尔的钙离子来诱导结合。这是由蛋白质不同的电荷所导致的。PS LUV对膜联蛋白V的结合能力在低pH值时更高。膜联蛋白V的结合会导致囊泡表面脱水以及双层膜内横向扩散的减少。虽然这种脱水在pH值为4时是由于囊泡接触引起的，但在pH值为7.4时是由于膜联蛋白V覆盖表面所致。膜联蛋白V在pH值为5及以下时促进LUV之间的磷脂混合，但在pH值为7.4时则抑制这种混合。用不带电荷的磷脂酰胆碱替代高达80%的PS并不影响pH值为4时磷脂混合的程度。PS LUV的高结合能力、抑制作用的消失以及计算得出的膜联蛋白V疏水性的增加使得膜联蛋白V在低pH值下能够穿透进入膜内成为可能。在中性pH值下，膜联蛋白V分子起到空间屏障的作用，阻止两个囊泡紧密靠近。在pH值为5时，膜联蛋白V降低了钙离子诱导的磷脂混合的阈值浓度。这种促进作用对于膜联蛋白VII（连丝蛋白）来说是众所周知的。这种效应可能与两种膜联蛋白的等电点有关，据报道它们的等电点分别为4.8（膜联蛋白V）和7.0（膜联蛋白VII）。
