Gregory Patrick, Ziemiecki Andrew, Zürcher Gisela, Brodbeck Urs, Bütikofer Peter
Institute of Biochemistry and Molecular Biology, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
Biol Chem. 2003 Dec;384(12):1575-82. doi: 10.1515/BC.2003.174.
Despite its well characterised biochemistry, the physiological role of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is unknown. Most of the previous studies investigating the distribution of GPI-PLD have focused on the human and bovine forms of the enzyme. Studies on mouse GPI-PLD are rare, partly due to the lack of a specific anti-mouse GPI-PLD antibody, but also due to the apparent low reactivity of existing antibodies to rodent GPI-PLDs. Here we describe the isolation of a mouse liver cDNA, the construction and expression of a recombinant enzyme and the generation of an affinity-purified rabbit anti-mouse GPI-PLD antiserum. The antibody shows good reactivity to partially purified murine and purified bovine GPI-PLD. In contrast, a rat anti-bovine GPI-PLD antibody shows no reactivity with the mouse enzyme and the two antibodies recognise different proteolytic fragments of the bovine enzyme. Comparison between the rodent, bovine and human enzymes indicates that small changes in the amino acid sequence of a short peptide in the mouse and bovine GPI-PLDs may contribute to the different reactivities of the two antisera. We discuss the implications of these results and stress the importance of antibody selection while investigating GPI-PLD in the mouse.
尽管糖基磷脂酰肌醇特异性磷脂酶D（GPI-PLD）的生物化学特性已得到充分表征，但其生理作用尚不清楚。以往大多数研究GPI-PLD分布的工作都集中在该酶的人类和牛类形式上。对小鼠GPI-PLD的研究很少，部分原因是缺乏特异性抗小鼠GPI-PLD抗体，也因为现有抗体对啮齿动物GPI-PLD的反应性明显较低。在此，我们描述了从小鼠肝脏中分离cDNA、构建和表达重组酶以及制备亲和纯化的兔抗小鼠GPI-PLD抗血清的过程。该抗体对部分纯化的小鼠GPI-PLD和纯化的牛GPI-PLD均表现出良好的反应性。相比之下，大鼠抗牛GPI-PLD抗体与小鼠酶无反应，且这两种抗体识别牛酶的不同蛋白水解片段。啮齿动物、牛和人类酶之间的比较表明，小鼠和牛GPI-PLD中短肽氨基酸序列的微小变化可能导致两种抗血清反应性不同。我们讨论了这些结果的意义，并强调在研究小鼠GPI-PLD时选择抗体的重要性。
