Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca(2+)-dependent membrane association.
Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca(2+)-dependent membrane association.
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来自人单核细胞的胞质磷脂酶 A2：底物特异性和 Ca(2 ) 依赖性膜关联的表征。
DOI：
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发表时间：
1993
影响因子：
4.1
通讯作者：
Margarete Goppelt
中科院分区：
文献类型：
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作者：
Woifgang Rehfeldt;Klaus Resch;Margarete Goppelt
通过离子交换、肝素-琼脂糖层析、疏水作用层析和非变性PAGE，从人单核细胞肿瘤细胞系THP-1中分离纯化出胞浆磷脂酶A2(PLA2)。纯化的PLA2在SDS/PAGE上的相对分子质量为100 kDa，而非变性PAGE的相对分子质量约为80 kDa。对PLA2底物专一性的研究表明，当使用标记的THP-1膜作为底物时，花生四烯酸没有优先于含有亚油酸的磷脂。用佛波酯佛波酯(PMA)处理THP-1细胞1~72h后，PMA刺激的THP-1细胞PLA2也得到了稳定的激活。比较部分纯化的PMA刺激细胞的PLA2和未刺激细胞的PLA2的性质。两种酶均表现出相同的钙依赖酶活性(EC50=300 nM钙离子)。两种来源的酶对Ca(2+)依赖的膜缔合作用有显著差异。与未经刺激的THP-1细胞的PLA2(300-500 nM)相比，经PMA刺激的细胞的PLA2在低钙浓度(100-300 nM)时显示出与PLA2的膜结合活性。这种钙依赖性的改变是由于酶的稳定修饰所致。因此，PLA2与细胞膜结合能力的变化可能被认为是激活细胞中观察到的脂肪酸代谢增加的重要分子机制。
A cytosolic phospholipase A2 (PLA2) has been purified to homogeneity from the human monocytic tumour cell line THP-1 by a combination of ion-exchange, heparin-agarose and hydrophobic-interaction chromatography and non-denaturing PAGE. The purified PLA2 exhibited a molecular mass of 100 kDa on SDS/PAGE, whereas non-denaturing PAGE showed a molecular mass of about 80 kDa. Investigation of the substrate specificity of PLA2 revealed no preference for arachidonic acid- over linoleic acid-containing phospholipids when labelled THP-1 membranes were used as substrate. Treatment of THP-1 cells for 1-72 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) led to a stable enzyme activation, which was also found after partial purification of PLA2 from PMA-stimulated THP-1 cells. The properties of the partially purified PLA2 from PMA-stimulated cells were compared with those of the PLA2 from unstimulated cells. Both enzymes showed an identical Ca(2+)-dependent enzyme activation (EC50 = 300 nM Ca2+). A significant difference was found in the Ca(2+)-dependent membrane association of the enzymes obtained from the two sources. PLA2 from PMA-stimulated cells showed membrane association of PLA2 activity at significantly lower Ca2+ concentrations (100-300 nM) compared with the PLA2 from unstimulated THP-1 cells (300-500 nM). The altered Ca(2+)-dependency was due to a stable modification of the enzyme. Changes in the ability of PLA2 to associate with cellular membranes may thus be regarded as an important molecular mechanism underlying the increase in fatty acid metabolism observed in activated cells.