Strum J C, Nixon A B, Daniel L W, Wykle R L
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1016.
Biochim Biophys Acta. 1993 Jul 21;1169(1):25-9. doi: 10.1016/0005-2760(93)90077-m.
A phosphono analog of choline phosphoglyceride was used to examine the relative contributions of phospholipase C and D in the generation of diglycerides in fMLP- and A23187-stimulated human neutrophils. The phosphono analog, 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphonocholine, contains a carbon-phosphorus bond adjacent to the base moiety and is resistant to phospholipase D hydrolysis, while remaining susceptible to phospholipase C hydrolysis. fMLP stimulated the production of [3H]phosphatidic acid and subsequently [3H]diglyceride from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine, but not from cells prelabeled with the phosphono analog. Treatment with A23187 also resulted in the formation of these products from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine. Additionally, A23187 stimulated the conversion of the phosphono analog to phosphodiester-containing choline phosphoglyceride which then resulted in the generation of [3H]phosphatidic acid and subsequently [3H]diglyceride. This study demonstrates the use of a phosphono analog in assessing phospholipase C and D activity in cells and provides evidence that in fMLP- and A23187-stimulated human neutrophils, diglyceride is generated indirectly from choline phosphoglycerides by the combined activities of phospholipase D and phosphatidate phosphohydrolase.
一种胆碱磷酸甘油酯的膦酸类似物被用于检测磷脂酶C和D在fMLP和A23187刺激的人中性粒细胞中甘油二酯生成过程中的相对贡献。该膦酸类似物，1-O-[³H]烷基-2-溶血-sn-甘油-3-膦酰胆碱，在碱基部分附近含有一个碳-磷键，对磷脂酶D水解具有抗性，而对磷脂酶C水解仍敏感。fMLP刺激含有1-O-[³H]烷基-2-酰基-sn-甘油-3-磷酸胆碱的细胞产生[³H]磷脂酸，随后产生[³H]甘油二酯，但不刺激预先用膦酸类似物标记的细胞。用A23187处理也导致含有1-O-[³H]烷基-2-酰基-sn-甘油-3-磷酸胆碱的细胞形成这些产物。此外，A23187刺激膦酸类似物转化为含磷酸二酯的胆碱磷酸甘油酯，然后导致[³H]磷脂酸的产生，随后是[³H]甘油二酯的产生。本研究证明了膦酸类似物在评估细胞中磷脂酶C和D活性方面的应用，并提供了证据表明，在fMLP和A23187刺激的人中性粒细胞中，甘油二酯是由磷脂酶D和磷脂酸磷酸水解酶的联合作用从胆碱磷酸甘油酯间接产生的。
