Raymond F D, Fortunato G, Moss D W
Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Clin Chim Acta. 1993 Jun 16;215(2):139-52. doi: 10.1016/0009-8981(93)90121-j.
A reproducible substrate for the assay of phosphatidylinositol-specific phospholipase D (PIPLD) can be prepared by extracting alkaline phosphatase from placental tissue with n-butanol under alkaline conditions. The alkaline phosphatase thus prepared retains its hydrophobic glycan phosphatidylinositol (GPI) anchor and aggregates into high M(r) forms. Incubation with serum hydrolyses the phosphate inositol linkage by PIPLD action, producing a less lipophilic, non-aggregated isoform of alkaline phosphatase. Three methods of measuring the amount of this isoform produced after a timed incubation with serum are described and compared: two types of phase partitioning systems, and electrophoresis and densitometry of the products after gradient-pore electrophoresis. All give comparable and reproducible measurements of PIPLD; however, the electrophoretic method is preferred for routine analysis.
用于磷脂酰肌醇特异性磷脂酶D（PIPLD）测定的可重现底物，可通过在碱性条件下用正丁醇从胎盘组织中提取碱性磷酸酶来制备。如此制备的碱性磷酸酶保留其疏水聚糖磷脂酰肌醇（GPI）锚，并聚集成高分子量形式。与血清一起温育通过PIPLD作用水解磷酸肌醇键，产生亲脂性较低的、非聚集的碱性磷酸酶同工型。描述并比较了三种在与血清定时温育后测量产生的这种同工型数量的方法：两种类型的相分配系统，以及梯度孔电泳后产物的电泳和光密度测定。所有方法对PIPLD的测量都具有可比性和可重复性；然而，电泳法更适合常规分析。
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