Pawelczyk T, Lowenstein J M
Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Biochem J. 1993 May 1;291 ( Pt 3)(Pt 3):693-6. doi: 10.1042/bj2910693.
Binding of phospholipase C delta 1 (PLC delta) to phospholipid vesicles was studied using large, unilamellar phospholipid vesicles (LUVs). PLC delta bound weakly to vesicles composed of phosphatidylserine (PS) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) + PC, and even more weakly to vesicles composed of phosphatidylinositol. The enzyme bound strongly to LUVs composed of PE + PC and phosphatidylinositol 4,5-bisphosphate (PIP2) or sphingomyelin (SM). Binding of 50% of PLC delta occurred at 0.25 nmol/ml PIP2 when LUVs composed of PE + PC (molar ratio of 80:20), plus various amounts of PIP2, were used at a constant phospholipid concentration of 300 nmol/ml. When LUVs composed of PE + PC + PIP2 (molar ratio of 79:20:1) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at 1.2 nmol/ml PIP2 and 120 nmol/ml total phospholipid. Similar measurements were conducted with other phospholipids and PIP2 at a molar ratio of 99:1. These showed that 50% binding of PLC delta occurred at a level of 0.9 nmol/ml PIP2 with 80 nmol/ml PC; at 2.2 nmol/ml PIP2 with 170 nmol/ml PS; at 4.2 nmol/ml PIP2 with 320 nmol/ml PI; and at 0.26 nmol/ml PIP2 with 20 nmol/ml total liver phospholipids. Binding to phosphatidylinositol 4-phosphate was much weaker. When LUVs composed of PE + PC + SM (molar ratio 48:12:40) were tested as a function of increasing phospholipid concentration, 50% binding of PLC delta occurred at a level of 96 nmol/ml SM. This is well below the concentration of SM that can be calculated to face the cytosol. Binding of PLC delta to LUVs decreased as the temperature was lowered from 37 degrees C to 0 degree C. Thus PLC delta shows a high degree of specificity for binding to PIP2 and SM. Under physiological conditions a considerable fraction of PLC delta may be bound to cellular membranes, either in an inactive form if bound to PIP2 at low resting Ca2+ concentrations, or in the inhibited form if bound to SM.
使用大单层磷脂囊泡（LUVs）研究了磷脂酶Cδ1（PLCδ）与磷脂囊泡的结合情况。PLCδ与由磷脂酰丝氨酸（PS）、磷脂酰胆碱（PC）或磷脂酰乙醇胺（PE）+PC组成的囊泡结合较弱，与由磷脂酰肌醇组成的囊泡结合更弱。该酶与由PE+PC和磷脂酰肌醇4,5-二磷酸（PIP2）或鞘磷脂（SM）组成的LUVs结合紧密。当使用由PE+PC（摩尔比80:20）组成的LUVs加上不同量的PIP2，在恒定磷脂浓度300 nmol/ml下时，50%的PLCδ结合发生在0.25 nmol/ml PIP2处。当测试由PE+PC+PIP2（摩尔比79:20:1）组成的LUVs随磷脂浓度增加的情况时，50%的PLCδ结合发生在1.2 nmol/ml PIP2和120 nmol/ml总磷脂处。以99:1的摩尔比用其他磷脂和PIP2进行了类似测量。结果表明，50%的PLCδ结合发生在以下水平：80 nmol/ml PC时为0.9 nmol/ml PIP2；170 nmol/ml PS时为2.2 nmol/ml PIP2；320 nmol/ml PI时为4.2 nmol/ml PIP2；20 nmol/ml总肝磷脂时为0.26 nmol/ml PIP2。与磷脂酰肌醇4-磷酸的结合要弱得多。当测试由PE+PC+SM（摩尔比48:12:40）组成的LUVs随磷脂浓度增加的情况时，50%的PLCδ结合发生在96 nmol/ml SM水平。这远低于可计算出的面向胞质溶胶的SM浓度。当温度从37℃降至0℃时，PLCδ与LUVs的结合减少。因此，PLCδ显示出对PIP2和SM结合的高度特异性。在生理条件下，相当一部分PLCδ可能与细胞膜结合，如果在低静息Ca2+浓度下与PIP2结合则以无活性形式存在，如果与SM结合则以受抑制形式存在。
