Lipoxygenation of arachidonic acid by subcellular preparations from murine keratinocytes.
Lipoxygenation of arachidonic acid by subcellular preparations from murine keratinocytes.
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鼠角质形成细胞的亚细胞制剂对花生四烯酸的脂氧合。
DOI：
10.1111/1523-1747.ep12340250
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Voorhees,JJ
中科院分区：
文献类型：
--
作者：
Ziboh,VA;Casebolt,TL;Marcelo,CL;Voorhees,JJ
在这些研究中，我们研究了小鼠角质形成细胞的无细胞制剂除了在这些细胞中建立花生四烯酸（AA）的环加氧酶途径外，还具有5-脂肪加氧酶活性的可能性。我们的数据表明，高速（105,000 g）小鼠角质形成细胞的上清制剂将[14C]AA代谢为标记的脂氧合酶产物。这些放射性代谢物的一部分分别在硅酸柱层析和薄层色谱（TLC）两种溶剂体系中与12-HETE（12-脂氧合酶途径标志物）和LTB4（5-脂氧合酶途径标志物）共层析和同步。通过反相（RP）和直相高压液相色谱分别与游离酸和正品ltb4共层析和甲酯共层析，进一步验证了与ltb4同源的novel14C在TLC和柱层析上的一致性。[14C]AA在ETYA、NDGA（环加氧酶和脂加氧酶途径抑制剂）和15-HETE（脂加氧酶途径抑制剂）存在下的无细胞制剂中孵育，导致[14C] 12-HETE和[14C] ltb4样代谢物的形成减少。相反，在吲哚美辛（一种环加氧酶抑制剂）存在的情况下，与[14C] AA一起培养无细胞提取物会导致标记的脂加氧酶代谢物的生物合成增加。这些数据表明，在小鼠角质形成细胞的可溶部分中存在酶，可以催化[14C] AA转化为12-和5-脂氧合酶途径的产物。
In these studies, we examined the possibility that cell-free preparations from murine keratinocytes possess 5-lipoxygenase activity in addition to the well-established cyclooxygenase pathway of arachidonic acid (AA) in these cells. Our data demonstrated that the high-speed (105,000 g) supernatant preparations of the murine keratinocytes metabolized [14C]AA into labeled lipoxygenase products. Portions of these radioactive metabolites cochromatographed and comigrated with 12-HETE (a marker for 12-lipoxygenase pathway) and with authentic LTB4(a marker for 5-lipoxygenase pathway) on silicic acid column chromatography and by thin-layer chromatography (TLC) in two solvent systems respectively. Identity of the novel14C which comigrated with LTB4on both TLC and column chromatography was verified further by cochromatography of the free acid with authentic LTB4on a reverse phase (RP) and the methyl esters on a straight phase high-pressure liquid chromatography. Incubation of the cell-free preparations with [14C]AA in the presence of ETYA, NDGA (inhibitors of cyclooxygenase and lipoxygenase pathways) as well as with 15-HETE (an inhibitor of lipoxygenase pathway) resulted in decreased formation of [14C] 12-HETE and the [14C]LTB4-like metabolite. On the contrary, incubations of the cell-free extracts with [14C] AA in the presence of indomethacin (a cyclooxygenase inhibitor) resulted in increased biosynthesis of the labeled lipoxygenase metabolites. These data indicate the existence of enzymes in soluble fraction of murine keratinocyte which can catalyze the transformation of [14C] AA into products of both the 12- and 5-lipoxygenase pathways.