影响血清中GPI-PLD活性检测的因素

Factors Affecting the Glycosylphosphatidylinositol-specific Phospholipase D Activity in Serum

采用在碱性条件下正丁醇抽提的人胎盘膜上的碱性磷酸酶（ＡＬＰ）作底物， 检测血清中糖基磷脂酰肌醇－ 特异性的磷脂酶Ｄ （ＧＰＩ－ＰＬＤ） 的活性水平． 这种ＡＬＰ 含疏水的ＧＰＩ锚定膜结构（ａｎｃｈｏｒ）， 与血清保温后能被其中的ＧＰＩ－ＰＬＤ降解成亲水的不含ＧＰＩ－锚定的ＡＬＰ． 采用Ｔｒｉｔｏｎ Ｘ－１１４ 二相分离法和梯度凝胶电泳法来分离含ＧＰＩ的ＡＬＰ和不含ＧＰＩ的ＡＬＰ， 计算出转化率（％ ）， 用来表示ＧＰＩ－ＰＬＤ酶活性． 对这两种方法进行比较后， 表明在一般实验室条件下， 二相分离法更为简便， 并对其影响因素进行了全面探讨．

A reproducible substrate for the assay of phosphatidylinositol specific phospho lipase D (GPI PLD) was prepared by extracting alkaline phosphatase (ALP) from human placental tissue with n butanol under alkaline conditions. The ALP thus retained its hydrophobic glycan phosphatidylinositol (GPI) anchor. Incubation with serum ALP was hydrolyzed the GPI linkage by GPI PLD action, producinga hydrophilic isoform. The degree of conversion of the two isoforms can be taken as a criterion for measuring GPI PLD activ ity of the serum sample. The results of two methods Triton X 114 phase partitioning system and gradient gel electrophoresis were compared, giving corresponding and reproducible GPI PLD activity in serum. However, the phase partitioning method is preferred forroutine analysis. Factors affecting the estimation are thoroughly discussed and the assay is reliable and sufficiently precise.