Moore R B, Appel S H
Can J Biochem Cell Biol. 1984 Jun;62(6):363-8. doi: 10.1139/o84-050.
Incubation of erythrocytes with [32P]phosphate resulted in a linear incorporation of the label into PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate), PtdIns4P (phosphatidylinositol 4-monophosphate), and PA (phosphatidic acid) over a period of 2 h at 37 degrees C. Exposure of 32P-labelled erythrocyte ghosts to calcium caused a loss of label from PtdIns(4,5)P2 and PtdIns4P, but not PA. The concentration of calcium required for half-maximal hydrolyses of both polyphosphoinositides was about 1 microM. Strontium, at higher concentrations, stimulated the hydrolyses of both polyphosphoinositides but barium, up to 1 mM, had little effect. Intact erythrocytes incubated in the presence of Ca-EGTA buffers and the ionophore A23187 did not show marked losses of [32P]PtdIns(4,5)P2 or [32P]PtdIns4P, but rather exhibited a dramatic increase in the level of [32P]PA. In contrast, cells which had been depleted of their ATP lost significant amounts of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P and had less change in their levels of [32P]PA relative to intact cells. The calcium activation curve and the time course for [32P]PA synthesis in intact cells were similar to the calcium activation curve and the time course for the hydrolyses of [32P]PtdIns(4,5)P2 and [32P]PtdIns4P in ATP-depleted erythrocytes. These results strongly support a link between Ca2+-dependent polyphosphoinositide breakdown and PA synthesis in human erythrocytes.
将红细胞与[32P]磷酸盐一起温育，在37℃下经过2小时，标记物呈线性掺入磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）、磷脂酰肌醇-4-单磷酸（PtdIns4P）和磷脂酸（PA）中。将32P标记的红细胞膜暴露于钙会导致PtdIns(4,5)P2和PtdIns4P中的标记物丢失，但PA不受影响。两种多磷酸肌醇半最大水解所需的钙浓度约为1微摩尔。较高浓度的锶刺激了两种多磷酸肌醇的水解，但高达1毫摩尔的钡几乎没有影响。在Ca-EGTA缓冲液和离子载体A23187存在下孵育的完整红细胞未显示出[32P]PtdIns(4,5)P2或[32P]PtdIns4P的明显丢失，而是表现出[32P]PA水平的显著增加。相比之下，ATP耗尽的细胞丢失了大量的[32P]PtdIns(4,5)P2和[32P]PtdIns4P，并且相对于完整细胞，其[32P]PA水平变化较小。完整细胞中[32P]PA合成的钙激活曲线和时间进程与ATP耗尽的红细胞中[32P]PtdIns(4,5)P2和[32P]PtdIns4P水解的钙激活曲线和时间进程相似。这些结果有力地支持了人红细胞中钙依赖性多磷酸肌醇分解与PA合成之间的联系。
