Protein kinase C as a measure of transbilayer phosphatidylserine asymmetry.
Protein kinase C as a measure of transbilayer phosphatidylserine asymmetry.
复制标题
蛋白激酶 C 作为跨双层磷脂酰丝氨酸不对称性的测量。
DOI：
10.1006/abio.1994.1080
复制
发表时间：
1994
影响因子：
2.9
通讯作者：
Newton,AC
中科院分区：
文献类型：
--
作者：
Daleke,DL;Huestis,WH;Newton,AC
正常红细胞的内源性磷脂酰丝氨酸局限于细胞膜的胞质小叶。然而，在病理条件下，跨膜不对称性可以改变，细胞表面的磷脂酰丝氨酸可能会出现在细胞表面。利用外源性蛋白激酶C的活化，建立了一种灵敏的测定红细胞膜磷脂酰丝氨酸含量的方法。含有外源性磷脂酰胆碱的红细胞纳入外膜单层不刺激蛋白激酶C活性比未经处理的细胞。相比之下，通过预先抑制氨基磷脂转运蛋白，将外源性磷脂酰丝氨酸掺入其膜外单层的红细胞刺激蛋白激酶C的作用显著高于允许外源性磷脂酰丝氨酸易位至内膜单层的红细胞。对于正常细胞和未暴露于脂质的细胞，激酶活化相当，其中氨基磷脂转运蛋白被巯基试剂（二酰胺或N-乙基马来酰亚胺）抑制。然而，Ca 2+负载导致蛋白激酶C的活化增加超过对照细胞，这与之前的报道一致，即Ca 2+诱导红细胞和血小板磷脂酰丝氨酸的暴露。通过参考模型系统中磷脂酰丝氨酸对蛋白激酶C的激活，可以估计细胞表面上磷脂酰丝氨酸的量。因此，蛋白激酶C的激活提供了对生物膜外单层中磷脂酰丝氨酸的灵敏且特异的测量。
The endogenous phosphatidylserine of normal erythrocytes is confined to the cytoplasmic leaflet of the membrane. However, under pathologic conditions transmembrane asymmetry can be altered and cytofacial phosphatidylserine may appear on the cell surface. A sensitive alternative method for the measurement of the exposed phosphatidylserine content of erythrocyte membranes was developed using the activation of exogenous protein kinase C. Erythrocytes containing exogenous phosphatidylcholine incorporated into the outer membrane monolayer do not stimulate protein kinase C activity more than untreated cells. In contrast, red cells that have exogenous phosphatidylserine incorporated into their membrane outer monolayer, by prior inhibition of the aminophospholipid transporter, stimulate protein kinase C significantly more than red cells in which exogenous phosphatidylserine is allowed to translocate to the inner membrane monolayer. Kinase activation is comparable for normal cells and cells not exposed to lipid in which the aminophospholipid transporter is inhibited with sulfhydryl reagents (diamide or N-ethylmaleimide). However, Ca2+-loading results in an increase in activation of protein kinase C over control cells, consistent with previous reports that Ca2+induces the exposure of erythrocyte and platelet phosphatidylserine. By reference to protein kinase C activation by phosphatidylserine in model systems, the quantity of phosphatidylserine on the cell surface may be estimated. Thus, protein kinase C activation affords a sensitive and specific measure of phosphatidylserine in the outer monolayer of biological membranes.