Horwitz J, Davis L L
Department of Pharmacology, Medical College of Pennsylvania, Philadelphia 19129.
Biochem J. 1993 Nov 1;295 ( Pt 3)(Pt 3):793-8. doi: 10.1042/bj2950793.
Neurotransmitters activate a phospholipase D that is though to specifically hydrolyse phosphatidylcholine. This enzyme has a unique property known as transphosphatidylation: in the presence of an appropriate nucleophilic receptor such as an alcohol, phospholipase D will catalyse the production of phosphatidyl-alcohol. We have studied phospholipase D using an in vitro assay that uses [3H]butanol of high specific radioactivity (15 Ci/mmol) as an acceptor. In the presence of [3H]butanol and phosphatidylcholine, a microsomal membrane fraction from rat brain catalysed the production of phosphatidyl[3H]butanol. Phospholipase D activity was dependent upon the presence of a detergent; the optimal sodium oleate concentration was between 4 and 6 mM. The RF of the phosphatidyl[3H]butanol on t.l.c. was identical to the RF of the phosphatidylbutanol formed when [3H]phosphatidylcholine was incubated with 100 mM butanol. These data confirm the identity of phosphatidyl[3H]butanol. One important advantage of this assay is that the substrate does not need to be labelled. We have used this advantage to examine the substrate specificity of phospholipase D. Microsomal phospholipase D appears to hydrolyse phosphatidylcholine most efficiently. There is a relatively small but significant activity against phosphatidylethanolamine and phosphatidylserine, and there is no significant activity against phosphatidylinositol. As the head-group becomes more like choline, the phospholipid becomes a better substrate for phospholipase D. The addition of one methyl group leads to a large increase in activity. Fatty acid composition does not play a role in determining the substrate specificity. This assay should be useful in furthering our understanding of this important enzyme.
神经递质激活一种磷脂酶D，据认为该酶能特异性水解磷脂酰胆碱。这种酶具有一种名为转磷脂酰基作用的独特性质：在存在合适的亲核受体如醇的情况下，磷脂酶D将催化磷脂酰醇的产生。我们使用一种体外测定法研究了磷脂酶D，该测定法使用高比放射性（15 Ci/mmol）的[3H]丁醇作为受体。在[3H]丁醇和磷脂酰胆碱存在的情况下，大鼠脑微粒体膜部分催化了磷脂酰[3H]丁醇的产生。磷脂酶D的活性依赖于去污剂的存在；油酸钠的最佳浓度在4至6 mM之间。磷脂酰[3H]丁醇在薄层层析上的Rf与[3H]磷脂酰胆碱与100 mM丁醇孵育时形成的磷脂酰丁醇的Rf相同。这些数据证实了磷脂酰[3H]丁醇的同一性。该测定法的一个重要优点是底物无需标记。我们利用这一优点研究了磷脂酶D的底物特异性。微粒体磷脂酶D似乎最有效地水解磷脂酰胆碱。对磷脂酰乙醇胺和磷脂酰丝氨酸有相对较小但显著的活性，而对磷脂酰肌醇没有显著活性。随着头部基团变得更像胆碱，磷脂成为磷脂酶D的更好底物。添加一个甲基会导致活性大幅增加。脂肪酸组成在决定底物特异性方面不起作用。该测定法应有助于增进我们对这种重要酶的理解。
