Fu Y, Cheng J X, Hong S L
Division of Cardiology, New England Deaconess Hospital, Boston, Massachusetts 02215.
Thromb Res. 1994 Mar 15;73(6):405-17. doi: 10.1016/0049-3848(94)90042-6.
Phospholipases C (PLCs) are ubiquitous enzymes which play key roles in the response of cells to extracellular agonists. Endothelial cells are involved in myriad normal and pathophysiologic functions. Although it is known that agonists activate PLCs in endothelial cells, second messengers form, and cellular responses ensue, more knowledge is needed about the specific types of PLCs in these cells. To this end, cytosolic PLCs from porcine aortic endothelial cells were partially purified by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B and heparin-agarose. Three PLC isozymes immunologically similar to bovine brain PLC-beta, PLC-gamma, and PLC-delta were identified. The relative levels of PLC activities in the cytosol were: PLC-beta, 50%; PLC-gamma, 44%; PLC-delta, 6%. The level of PLC-beta activity in porcine endothelial cells appeared higher than the levels reported for several established cell lines, suggesting that this enzyme may play a specific role in endothelial cell function. Elution profiles of PLC activity with phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) as substrate were similar to those with phosphatidylinositol (Ptdlns) as substrate, indicating that cytosolic PLCs hydrolyze both Ptdlns and Ptdlns(4,5)P2 and no Ptdlns(4,5)P2-specific PLC was present in the cytosol. The catalytic properties of the partially purified PLC isozymes from porcine endothelial cells were similar to their counterparts from bovine brain. These include the dependence of hydrolysis of Ptdlns on Ca2+, the optimal Ca2+ concentrations for the hydrolysis of Ptdlns and Ptdlns(4,5)P2, the pH optima, and the stimulatory effects of deoxycholate.
磷脂酶C（PLCs）是普遍存在的酶，在细胞对细胞外激动剂的反应中起关键作用。内皮细胞参与众多正常和病理生理功能。虽然已知激动剂可激活内皮细胞中的PLCs，形成第二信使并引发细胞反应，但对于这些细胞中PLCs的具体类型仍需更多了解。为此，通过硫酸铵分级分离以及在DEAE-琼脂糖凝胶CL-6B和肝素-琼脂糖上进行柱色谱，对猪主动脉内皮细胞的胞质PLCs进行了部分纯化。鉴定出三种免疫上与牛脑PLC-β、PLC-γ和PLC-δ相似的PLC同工酶。胞质中PLC活性的相对水平为：PLC-β，50%；PLC-γ，44%；PLC-δ，6%。猪内皮细胞中PLC-β的活性水平似乎高于几种已建立细胞系所报道的水平，这表明该酶可能在内皮细胞功能中发挥特定作用。以磷脂酰肌醇4,5-二磷酸（Ptdlns(4,5)P2）为底物时PLC活性的洗脱曲线与以磷脂酰肌醇（Ptdlns）为底物时相似，表明胞质PLCs可水解Ptdlns和Ptdlns(4,5)P2，且胞质中不存在Ptdlns(4,5)P2特异性PLC。从猪内皮细胞中部分纯化的PLC同工酶的催化特性与其牛脑对应物相似。这些特性包括Ptdlns水解对Ca2+的依赖性、Ptdlns和Ptdlns(4,5)P2水解的最佳Ca2+浓度、最适pH以及脱氧胆酸盐的刺激作用。
