Bunt G, de Wit J, van den Bosch H, Verkleij A J, Boonstra J
Department of Molecular Cell Biology, Utrecht University, Utrecht, The Netherlands.
J Cell Sci. 1997 Oct;110 ( Pt 19):2449-59. doi: 10.1242/jcs.110.19.2449.
The 85 kDa cytosolic phospholipase A2 is the key enzyme in the release of arachidonic acid. To gain insight into cytosolic phospholipase A2 action in mitogen-activated cells, the localization of the phospholipase was investigated in fibroblasts upon stimulation with epidermal growth factor and the calcium ionophore A23187. By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phospholipase A2 resulted in a punctate labeling pattern randomly distributed throughout the cytoplasm of the cell. Immunogold electron microscopy revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A2 in small clusters. These clusters were found in the cytosol in the vicinity of all organellar membranes, except for the Golgi system. The enzyme showed no preference for the nuclear envelope, the endoplasmic reticulum or the plasma membrane. Stimulation of cells with epidermal growth factor or A23187 or both did not change the punctate immunofluorescence labeling pattern. Furthermore, a similar labeling pattern was observed by the artificial introduction of extremely low or high intracellular calcium concentrations. Even by electron microscopy, translocation of cytosolic phospholipase A2 to membranes was not observed after stimulation of cells with epidermal growth factor and A23187. From these results it is concluded that cytosolic phospholipase A2 is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.
85 kDa的胞质型磷脂酶A2是花生四烯酸释放过程中的关键酶。为深入了解有丝分裂原激活细胞中胞质型磷脂酶A2的作用，研究了在表皮生长因子和钙离子载体A23187刺激下成纤维细胞中该磷脂酶的定位情况。通过间接免疫荧光显微镜观察，内源性胞质型磷脂酶A2的染色呈现出点状标记模式，随机分布于整个细胞质中。免疫金电子显微镜显示，这种点状标记模式表明85 kDa的磷脂酶A2以小簇形式存在。这些小簇存在于除高尔基体系统外的所有细胞器膜附近的胞质溶胶中。该酶对核膜、内质网或质膜没有偏好。用表皮生长因子或A23187或两者刺激细胞均未改变点状免疫荧光标记模式。此外，通过人为引入极低或极高的细胞内钙浓度也观察到了类似的标记模式。即使通过电子显微镜观察，在用表皮生长因子和A23187刺激细胞后，也未观察到胞质型磷脂酶A2向膜的转位。从这些结果可以得出结论，在受刺激和未受刺激的成纤维细胞中，胞质型磷脂酶A2均定位于靠近膜的小簇中，对特定的细胞器膜没有偏好。
