Mackin W M, Stevens T M
Medical Products Department, E.I. DuPont de Nemours & Co., Inc., Wilmington, Delaware.
J Leukoc Biol. 1988 Jul;44(1):8-16. doi: 10.1002/jlb.44.1.8.
The phosphatidylinositol (PI)-specific phospholipase C (PLC) activity contained in sonicates of casein-elicited (4-6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of PI by the sonicate PLC is linear for 15-20 min at 37 degrees C and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5-6.0, is enhanced 1.5-3-fold by the addition of deoxycholate, and is Ca++ dependent. Kinetic analysis of the PLC hydrolysis of PI yields an apparent Km of 240 +/- 85 microM and a Vmax of 34.3 +/- 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 +/- 66 microM and a Vmax of 14.3 +/- 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to PI. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values less than or equal to 100 microM) inhibitors of the PMN PLC.
已对酪蛋白诱导（4 - 6小时）的大鼠中性粒细胞超声裂解液中的磷脂酰肌醇（PI）特异性磷脂酶C（PLC）活性进行了鉴定和表征。以磷脂酰肌醇（PI）为底物时，在中性粒细胞超声裂解液100,000g离心的上清液和沉淀中均发现了PLC活性。通过在不连续蔗糖梯度上离心对粗超声裂解液进行进一步分级分离表明，PLC活性主要存在于胞质溶胶中，在富含质膜和颗粒的级分中活性较少。超声裂解液PLC对PI的水解在37℃下15 - 20分钟内呈线性，并且相对于添加的超声裂解液蛋白量也是线性的。该酶对PI具有选择性，对其他磷脂如磷脂酰乙醇胺（PE）、磷脂酰丝氨酸（PS）或磷脂酰胆碱（PC）几乎没有水解活性。PLC活性的最适pH为5.5 - 6.0，添加脱氧胆酸盐可使其活性提高1.5 - 3倍，并且依赖于Ca++。对PI的PLC水解进行动力学分析得出表观Km为240±8 microM，Vmax为34.3±17.0 nmol/min/mg蛋白（n = 3）。同样，当使用磷脂酰肌醇4,5 - 二磷酸（PIP2）作为底物时，得到表观Km为109±66 microM，Vmax为14.3±10.4 nmol/min/mg（n = 3）蛋白。这些数据表明，相对于PI，PIP2可能是PMN PLC的稍好底物。最后，测试了先前报道在体外抑制血小板PLC活性的多种药物抑制大鼠PMN PLC的能力。在所测试的化合物中，没有一种是PMN PLC的强效（即IC5值小于或等于100 microM）抑制剂。
