Matsuda R, Kaneko N, Horikawa Y
Department of Anatomy, Tokyo Women's Medical College, Japan.
Biochem Biophys Res Commun. 1997 Aug 28;237(3):499-503. doi: 10.1006/bbrc.1997.7177.
This study evaluated whether annexins I, II and VI possess Ca2+ transport activity in phospholipid membranes by the burst method, and the activity of each was compared with that of annexin V. Briefly, in the presence of 400 microM Ca2+, each annexin at 50 nM was added to large unilamellar vesicles (LUV) which were then burst in fura-2 solution with 0.2% Triton X-100, followed by examination of Ca2+ signals. Annexins I, II, V and VI were all shown to express, each to a different degree, Ca2+ activity toward phosphatidylserine/phosphatidyl- ethanolamine-LUV. Ca2+ signal intensity increased dependent on annexin concentration, and the Ca2+ transport activity of annexin V and VI was higher than that of annexin I and II. However, none of annexin I, II, V and VI expressed Ca2+ transport activity in LUV produced using phosphatidylcholine. Ca(2+)-incorporated LUV with no annexin showed signals whose intensity was proportional to Ca2+ concentration. The Ca2+ transport activity of the annexins could be effectively measured by the burst method. Ca2+ signal intensity would thus appear to be unique for each of the annexins and to be determined by the particular function and specificity of each of those considered in this study.
本研究采用破裂法评估膜联蛋白I、II和VI在磷脂膜中是否具有Ca2+转运活性，并将它们各自的活性与膜联蛋白V进行比较。简要地说，在存在400微摩尔Ca2+的情况下，将50纳摩尔的每种膜联蛋白添加到大单层囊泡（LUV）中，然后在含有0.2% Triton X-100的fura-2溶液中使囊泡破裂，随后检测Ca2+信号。结果显示，膜联蛋白I、II、V和VI均对磷脂酰丝氨酸/磷脂酰乙醇胺-LUV表现出不同程度的Ca2+活性。Ca2+信号强度随膜联蛋白浓度增加而增强，且膜联蛋白V和VI的Ca2+转运活性高于膜联蛋白I和II。然而，膜联蛋白I、II、V和VI在由磷脂酰胆碱制备的LUV中均未表现出Ca2+转运活性。不含膜联蛋白的Ca(2+)-掺入LUV显示出强度与Ca2+浓度成正比的信号。通过破裂法能够有效地测定膜联蛋白的Ca2+转运活性。因此，Ca2+信号强度对于每种膜联蛋白而言似乎是独特的，并且由本研究中所考虑的每种膜联蛋白的特定功能和特异性所决定。
