Birmelin M, Marme D, Ferber E, Decker K
Eur J Biochem. 1984 Apr 2;140(1):55-61. doi: 10.1111/j.1432-1033.1984.tb08066.x.
A protein resembling calmodulin was isolated from non-parenchymal and parenchymal cells of rat liver by affinity chromatography. The biological activity of the purified protein was assessed by the bovine brain cAMP phosphodiesterase assay. A highly sensitive radioimmunoassay as well as the cAMP phosphodiesterase method were employed to determine the calmodulin content of crude extracts from monolayer cultures of rat Kupffer cells and hepatocytes. An ATP-dependent, calmodulin-enhanced calcium transport was demonstrated in a membrane fraction of the non-parenchymal cells. Phospholipase A2 activity specific for 2-arachidonoyl phosphatide and with a pH optimum of 8.1 was measured in homogenized Kupffer cells; it was stimulated by agents previously shown to enhance prostaglandin synthesis in Kupffer cells, e.g. zymosan particles and lipopolysaccharide isolated from Salmonella minnesota. The increase in activity was completely prevented by pretreatment with or simultaneous addition of R 24571, a known calmodulin antagonist. However, if this inhibitor or calmodulin was added to the cell-free extract phospholipase A2 activity was not influenced. Phospholipase A1 activity could be detected at pH 5 only, showing a slight decrease in the homogenate of stimulated macrophages. Acyltransferase activity was high but independent of treatment of the Kupffer cells.
通过亲和层析从大鼠肝脏的非实质细胞和实质细胞中分离出一种类似钙调蛋白的蛋白质。通过牛脑环磷酸腺苷磷酸二酯酶测定法评估纯化蛋白质的生物活性。采用高灵敏度放射免疫测定法以及环磷酸腺苷磷酸二酯酶法测定大鼠库普弗细胞和肝细胞单层培养物粗提物中的钙调蛋白含量。在非实质细胞的膜部分证实了一种依赖三磷酸腺苷、钙调蛋白增强的钙转运。在匀浆的库普弗细胞中测量了对2-花生四烯酰磷脂具有特异性且最适pH为8.1的磷脂酶A2活性；它受到先前显示能增强库普弗细胞中前列腺素合成的试剂的刺激，例如从明尼苏达沙门氏菌分离的酵母聚糖颗粒和脂多糖。用已知的钙调蛋白拮抗剂R 24571预处理或同时添加可完全阻止活性增加。然而，如果将这种抑制剂或钙调蛋白添加到无细胞提取物中，磷脂酶A2活性不受影响。磷脂酶A1活性仅在pH 5时可检测到，在受刺激巨噬细胞的匀浆中略有下降。酰基转移酶活性很高，但与库普弗细胞的处理无关。
