Boustead C M, Brown R, Walker J H
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):601-8. doi: 10.1042/bj2910601.
Annexin V has been purified from chicken liver; 40 mg of annexin V was obtained per kg of tissue. In contrast with mammalian liver, very little annexin VI was obtained. Surprisingly, chicken liver annexin V resembles mammalian annexin IV in its M(r) (32,500) and its isoelectric point (5.6), but amino-acid-sequence analysis demonstrates identity with chicken annexin V (anchorin CII). It binds to phospholipids in a Ca(2+)-dependent manner with free-Ca2+ concentrations for half-maximal binding to phosphatidylserine and phosphatidic acid of 10 microM; phosphatidylethanolamine of 32 microM and phosphatidylinositol of 90 microM. No binding to phosphatidylcholine was observed at Ca2+ concentrations up to 300 microM. In isolated liver membranes a significant proportion of annexin V was not extractable with EGTA but could only be extracted with Triton X-100, suggesting the existence of a tightly membrane-associated form of annexin V. A specific antiserum to chicken annexin V was used to localize the protein in adult and embryonic chicken liver. In the adult, annexin V was highly concentrated in epithelial cells lining the bile ducts, and along the bile canaliculi. In embryonic liver, strong staining of the bile-duct epithelial cells was again evident, and in addition, endothelial cells were strongly immunoreactive.
膜联蛋白V已从鸡肝中纯化出来；每千克组织可获得40毫克膜联蛋白V。与哺乳动物肝脏不同的是，膜联蛋白VI的产量非常少。令人惊讶的是，鸡肝膜联蛋白V在其相对分子质量（32,500）和等电点（5.6）方面与哺乳动物膜联蛋白IV相似，但氨基酸序列分析表明它与鸡膜联蛋白V（锚定蛋白CII）相同。它以Ca(2+)依赖的方式与磷脂结合，与磷脂酰丝氨酸和磷脂酸半最大结合时的游离Ca2+浓度为10微摩尔；与磷脂酰乙醇胺结合时为32微摩尔，与磷脂酰肌醇结合时为90微摩尔。在Ca2+浓度高达300微摩尔时，未观察到与磷脂酰胆碱的结合。在分离的肝细胞膜中，相当一部分膜联蛋白V不能用乙二醇双四乙酸（EGTA）提取，而只能用曲拉通X-100提取，这表明存在一种与膜紧密结合的膜联蛋白V形式。一种针对鸡膜联蛋白V的特异性抗血清被用于在成年和胚胎鸡肝中定位该蛋白。在成年鸡肝中，膜联蛋白V高度集中在胆管内衬的上皮细胞以及胆小管周围。在胚胎肝中，胆管上皮细胞再次出现强烈染色，此外，内皮细胞也有强烈的免疫反应。
