Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study.
Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study.
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来自蜡样芽孢杆菌的磷脂酰肌醇特异性磷脂酶 C 结合了内在磷酸转移酶和环状磷酸二酯酶活性：31P NMR 研究。
DOI：
10.1021/bi00487a010
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Griffith,OH
中科院分区：
文献类型：
--
作者：
Volwerk,JJ;Shashidhar,MS;Kuppe,A;Griffith,OH
1990年5月14日收到的修订版Mannipt摘要：通过31 P NMR分析蜡状芽孢杆菌的β-磷脂酰肌醇特异性磷脂酶C裂解磷脂酰肌醇期间形成的磷酸肌醇产物。31 P NMR光谱可以区分肌醇磷酸物质和磷脂酰肌醇。对于α-磷脂酰肌醇、肌醇1-单磷酸、肌醇2-单磷酸和肌醇1，2-环单磷酸，观察到的化学位移值（相对于磷酸）分别为-0.41、3.62、4.45和16.30 ppm。在各种实验条件下，该磷脂酶C通过分子内磷酸转移反应裂解磷脂酰肌醇，生成甘油二酯和D-肌醇1，2-环一磷酸。我们还报告了新的和意想不到的观察，磷脂酰肌醇特异性磷脂酶C从B。蜡状芽孢杆菌能够水解肌醇环磷酸以形成D-肌醇1-单磷酸。因此，该酶具有磷酸转移酶和环磷酸二酯酶活性。第二个反应需要千倍更高的酶浓度，以观察31 P NMR。该反应被证明是区域特异性的，因为仅产生1-磷酸，并且立体特异性的是，仅D-肌醇1，2-环一磷酸被水解。用对B具有特异性的单克隆抗体进行抑制。蜡状芽孢杆菌磷脂酶C表明，环状磷酸二酯酶活性是细菌酶固有的。我们提出了来自B的磷脂酰肌醇特异性磷脂酶C的两步机制。蜡状芽孢杆菌涉及连续的磷酸转移酶和环状磷酸二酯酶活性。这种机制与胰腺核糖核酸酶RNase A的两步机制相似。磷脂酰肌醇特异性磷脂酶C（PI-PLC）1（EC 3.1. 4.10）催化膜脂磷脂酰肌醇（Ptdlns）的裂解，产生膜溶性产物二酰基甘油和水溶性产物磷酸肌醇。在哺乳动物细胞中，PI-PLC作为具有有限结构同源性的蛋白质家族出现（Rhee等人，1989年）。这些
Revised Manuscript Received May 14, 1990 abstract: The inositol phosphate products formed during the cleavage of phosphatidylinositol by phos-phatidylinositol-specific phospholipase C from Bacillus cereus were analyzed by 31P NMR. 31P NMR spectroscopy can distinguish between the inositol phosphate species and phosphatidylinositol. Chemical shift values (with reference to phosphoric acid) observed are-0.41, 3.62, 4.45, and 16.30 ppm for phos-phatidylinositol, myo-inositol 1-monophosphate, myoinositol 2-monophosphate, and myoinositol 1, 2-cyclic monophosphate, respectively. It is shown that under a variety of experimental conditions thisphospholipase C cleaves phosphatidylinositol via an intramolecular phosphotransfer reaction producing diacylglycerol and D-myoinositol 1, 2-cyclicmonophosphate. We also report the new and unexpected observation that the phosphatidylinositol-specific phospholipase C from B. cereus is able to hydrolyze the inositol cyclic phosphate to form D-myoinositol 1-monophosphate. The enzyme, therefore, possesses phosphotransferase and cyclic phosphodiesterase activities. The second reaction requires thousandfold higher enzyme concentrations to be observed by 31P NMR. This reaction was shown to be regiospecific in that only the 1-phosphate was produced and stereospecific in that only D-myoinositol 1, 2-cyclic monophosphate was hydrolyzed. Inhibition with a monoclonal antibody specific for the B. cereus phospholipase C showed that the cyclic phosphodiesterase activity is intrinsic to the bacterial enzyme. We propose a two-step mechanism for the phosphatidylinositol-specific phospholipase C from B. cereus involving sequential phosphotransferase and cyclic phos-phodiesterase activities. This mechanism bears a resemblance to the well-known two-step mechanism of pancreatic ribonuclease, RNase A.Xhosphatidylinositol-specificphospholipaseC (PI-PLC) 1 (EC 3.1. 4.10) catalyzes cleavage of the membrane lipid phospha-tidylinositol (Ptdlns), producing a membrane-soluble product, diacylglycerol, and a water-soluble product, inositol phosphate. In mammalian cells, PI-PLC occurs as a family of proteins with limited structural homology (Rhee et al., 1989). These