Matecki A, Pawelczyk T
Department of Clinical Biochemistry, Medical University of Gdansk, Poland.
Biochim Biophys Acta. 1997 Apr 26;1325(2):287-96. doi: 10.1016/s0005-2736(96)00267-2.
Sphingosine, which is on the pathway of sphingomyelin degradation, activates phospholipase C (PLC) delta1 moderately. In the liposome assay effect of sphingosine on PLC delta1 activity depends on KCl concentration. Stimulation of PLC delta1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhibition of PLC delta1 by sphingomyelin. To determine the domain of PLC delta1 which interacts with sphingosine active proteolytic fragments of PLC delta1 were generated by trypsin digestion of the native enzyme. Sphingosine affects the activity of PLC delta1 fragment which lacked the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of PLC delta1. These observations indicate that for interaction of sphingosine with PLC delta1 intact domain that span regions of conservation, designated as X and Y is necessary. When the activity of PLC delta1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphingosine strongly inhibited PLC delta1. The other homolog of sphingosine 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta1 to much lesser extent. The activity of PLC delta1 was inhibited by 68% and 22% in the presence of 20 microM sphingosine and phytosphingosine, respectively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta1 in the cell.
鞘氨醇处于鞘磷脂降解途径中，可适度激活磷脂酶C（PLC）δ1。在脂质体测定中，鞘氨醇对PLCδ1活性的影响取决于氯化钾浓度。随着氯化钾浓度从0增加到100 mM，鞘氨醇对PLCδ1的刺激作用增强，然后随着氯化钾浓度的增加而减弱。在脂质体测定中，鞘氨醇可减轻鞘磷脂对PLCδ1的抑制作用。为了确定PLCδ1与鞘氨醇相互作用的结构域，通过用胰蛋白酶消化天然酶产生了PLCδ1的活性蛋白水解片段。鞘氨醇影响缺乏氨基末端结构域（前60个氨基酸）的PLCδ1片段的活性，但不影响已切割跨越PLCδ部分X和Y区域结构域的活性片段。这些观察结果表明，鞘氨醇与PLCδ1相互作用需要完整的、跨越保守区域（称为X和Y）的结构域。当以红细胞膜中的磷脂酰肌醇-4,5-二磷酸（PIP2）为底物测定PLCδ1的活性时，鞘氨醇强烈抑制PLCδ1。鞘氨醇的另一个同系物4-羟基鞘氨醇（植物鞘氨醇）对PLCδ1的抑制作用要小得多。在存在20 μM鞘氨醇和植物鞘氨醇的情况下，PLCδ1的活性分别被抑制68%和22%。在浓度为1.5 mM的脱氧胆酸盐存在下，这种抑制作用完全消除。这些观察结果表明，鞘氨醇可能在细胞中调节PLCδ1的活性。
