Kihn L, Rutkowski D, Stinson R A
Department of Pathology, University of Alberta, Edmonton, Canada.
Biochem Cell Biol. 1990 Sep;68(9):1112-8. doi: 10.1139/o90-166.
As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.
通过掺入脂质体和吸附到辛基琼脂糖上评估，来自人肝脏和胎盘的纯化四聚体形式碱性磷酸酶的膜锚完整性完好。任何导致二聚体酶的处理都排除了掺入和吸附。完整的锚也允许掺入红细胞血影。添加疏水蛋白会不同程度地抑制掺入脂质体。碱性磷酸酶可被对磷脂酰肌醇具有特异性的磷脂酶C从脂质体和红细胞血影中100%释放出来。胎盘碱性磷酸酶和肝脏碱性磷酸酶的释放速率没有明显差异，尽管两者从脂质体中的释放速度比从培养的骨肉瘤细胞悬液中释放慢约250倍，从红细胞血影中释放慢约100倍。两种酶都以二聚体形式被磷脂酰肌醇磷脂酶C释放，并且不会重新掺入或吸附到辛基琼脂糖上。然而，通过梯度凝胶电泳，掺入、用Triton X-100再溶解并用丁醇处理去除去污剂后的酶是四聚体，具有疏水性，并且可以重新掺入新鲜脂质体中。一种针对肝脏碱性磷酸酶的单克隆抗体抑制该酶掺入脂质体，并消除其从脂质体中的释放以及通过磷脂酰肌醇磷脂酶C转化为二聚体。
