Pawelczyk T, Matecki A
Department of Clinical Biochemistry, Medical University of Gdansk, Poland.
Eur J Biochem. 1998 Oct 1;257(1):169-77. doi: 10.1046/j.1432-1327.1998.2570169.x.
The localization of phospholipase C delta3 (PLC delta3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC delta3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC delta3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC delta3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC delta1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC delta1 is activated by spermine and sphingosine, whereas PLC delta3 was inhibited by both these compounds at concentrations that maximally stimulated PLC delta1. A deletion mutant of PLC delta3, lacking the entire pleckstrin homology (PH) domain (residues 1-137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC delta3 activation required calcium ions. The relationship between the Ca2+ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC delta3 was less sensitive to Ca2+ stimulation. This is in contrast to PLC delta1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposome assays. We conclude that Ca2+ is necessary to induce specific conformational changes of PLC delta3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC delta3 described in this report suggest that PLC delta3 has a relatively low activity in cellular conditions that fully activate PLC delta1.
对磷脂酶Cδ3（PLCδ3）在细胞中的定位及其调节特性进行了研究。蛋白质印迹法显示，人血小板PLCδ3位于膜和胞质组分中。胞质组分中的酶量明显低于膜组分中的酶量。在大鼠肝脏中，PLCδ3存在于膜和胞质组分中，细胞核中不存在。对磷脂对PLCδ3作用的研究表明，该酶受到磷脂酰乙醇胺（PtdEtn）和磷脂酰胆碱（PtdCho）的抑制。在鞘磷脂和磷脂酰丝氨酸（PtdSer）存在的情况下也观察到类似的抑制作用。这与PLCδ1相反，PLCδ1被PtdCho和PtdEtn激活。在去污剂测定中，PLCδ1被精胺和鞘氨醇激活，而PLCδ3在最大程度刺激PLCδ1的浓度下被这两种化合物抑制。PLCδ3的一个缺失突变体，缺少整个普列克底物蛋白同源（PH）结构域（第1 - 137位氨基酸残基），在去污剂测定中具有完全活性，并且与天然酶一样受到精胺、鞘氨醇和磷脂的同等程度抑制。PLCδ3的激活需要钙离子。缺失突变体和天然酶的Ca2 +浓度与酶活性之间的关系几乎相同。然而，在脂质体测定中，PLCδ3对Ca2 +刺激的敏感性较低。这与PLCδ1相反，PLCδ1在去污剂和脂质体测定中对Ca2 +刺激的敏感性相同。我们得出结论，Ca2 +是诱导PLCδ3特定构象变化所必需的，这导致催化结构域相对于膜的有效取向。本报告中描述的PLCδ3的调节特性表明，在完全激活PLCδ1的细胞条件下，PLCδ3具有相对较低的活性。
