Determination of the substrate specificity of the phospholipase D from Streptomyces chromofuscus via an inorganic phosphate quantitation assay.
Determination of the substrate specificity of the phospholipase D from Streptomyces chromofuscus via an inorganic phosphate quantitation assay.
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DOI：
10.1006/abio.1999.4420
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发表时间：
2000-02
影响因子：
2.9
通讯作者：
S. F. Martín;R. DeBlanc;P. Hergenrother
中科院分区：
文献类型：
--
作者：
S. F. Martín;R. DeBlanc;P. Hergenrother
利用基于无机磷酸盐定量的测定方法，测定了色褐链霉菌磷脂酶D（PLD（Sc））的底物特异性。以1，2-二-正己酰磷脂酰胆碱（C6 PC）、磷脂酰乙醇胺（C6 PE）、磷脂酰丝氨酸（C6 PS）、磷脂酰甘油（C6 PG）和带有新己基头基的非天然磷脂（C6 PDB）为底物进行检测。该测定依赖于用EDTA猝灭PLD（Sc）催化的磷脂底物水解，然后用碱性磷酸酶水解磷脂酸产物。由此释放的无机磷酸盐通过与碳酸铵形成络合物来定量，该络合物在700 nm处具有最大吸光度。为了最小化所涉及的时间和消耗的试剂，在96孔板中进行测定。结果表明，PLD（Sc）在底物上的催化效率为C6 PC>> C6 PS，约为C6 PE> C6 PG>> C6 PDB。
The substrate specificity for phospholipase D from Streptomyces chromofuscus (PLD(Sc)) has been determined utilizing an assay based on the quantitation of inorganic phosphate. 1,2-Di-n-hexanoyl phosphatidylcholine (C6PC), phosphatidylethanolamine (C6PE), phosphatidylserine (C6PS), phosphatidylglycerol (C6PG), and an unnatural phospholipid bearing a neohexyl headgroup (C6PDB) were examined as substrates. The assay relies on the quenching of the PLD(Sc)-catalyzed hydrolysis of the phospholipid substrates with EDTA followed by the hydrolysis of the phosphatidic acid product with alkaline phosphatase. The inorganic phosphate thus released is quantitated through the formation of a complex with ammonium molybdate, which has an absorbance maximum at 700 nm. To minimize the time involved and the reagents consumed, the assay is conducted in 96-well plates. The results of this study indicate that the catalytic efficiency for PLD(Sc) on the substrates is C6PC >> C6PS approximately C6PE > C6PG >> C6PDB.