ARACHIDONATE-CONTAINING TRIACYLGLYCEROLS - BIOSYNTHESIS AND A LIPOLYTIC MECHANISM FOR THE RELEASE AND TRANSFER OF ARACHIDONATE TO PHOSPHOLIPIDS IN HL-60 CELLS
ARACHIDONATE-CONTAINING TRIACYLGLYCEROLS - BIOSYNTHESIS AND A LIPOLYTIC MECHANISM FOR THE RELEASE AND TRANSFER OF ARACHIDONATE TO PHOSPHOLIPIDS IN HL-60 CELLS
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DOI：
10.1016/0005-2760(93)90010-7
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发表时间：
1993-11-03
期刊：
影响因子：
--
通讯作者：
SNYDER, F
中科院分区：
文献类型：
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作者：
BLANK, ML;SMITH, ZL;SNYDER, F
当HL-60细胞在含有10 muM [H-3]花生四烯酸的培养基中孵育时，标签立即掺入甘油三酯和磷脂中。孵育2小时后，大约一半的细胞氚与三酰基甘油相关，一旦将标记的细胞置于不含花生四烯酸的培养基中，这种[h -3]花生四烯酸就被转移到磷脂中。设计了一种技术来分析[H-3]花生四烯酸酯在甘油的三个sn位置的立体特异性分布，以确定标记的三酰基甘油的生物合成机制。[H-3]花生四烯酸酯在三酰基甘油的所有三个甘油位中分布的量几乎相等。此外，对含有[H-3]花生四烯酸酯的完整三酰基甘油的分析表明，用反相高效液相色谱法洗脱了24%的氚。如果含有花生四烯酸酯的三酰基甘油的很大一部分是从头合成的，那么这两个发现都是意料之中的。由[H-3]花生四烯酸预标记的HL-60细胞制备的浆液能够水解内源性[H-3]花生四烯酸三酰基甘油，主要产生游离脂肪酸和少量单酰基甘油。匀浆的脂解活性产生了相对少量的单酰基甘油和二酰基甘油，这表明[H-3]花生四烯酸酯是由[H-3]三酰基甘油的所有三个sn位水解而成的。脂肪酶活性的最适pH值为4.5，与可溶性组分的关系大于与总膜组分的关系。虽然尚不清楚这种脂溶活性是否与完整细胞中表达的相同，但在将完整的HL-60细胞（预标记为[H-3]花生四烯酸酯）转移到新鲜培养基后观察到的[H-3]三酰甘油脂肪酶的活性足够大，可以很容易地解释[H-3]三酰甘油的减少。数据表明花生四烯酸从三酰基甘油到磷脂的转移可能是通过利用溶血磷脂和花生四烯醇辅酶a的酰基转移酶进行的。
When HL-60 cells are incubated in media containing 10 muM [H-3]arachidonic acid the label is immediately incorporated into both triacylglycerols and phospholipids. About one-half of the cellular tritium was associated with triacylglycerols after 2 h of incubation and this [H-3]arachidonate was then transferred to phospholipids as soon as the labeled cells were placed in arachidonate-free media. A technique was devised to analyze the stereospecific distribution of [H-3]arachidonate at the three sn-positions of glycerol in order to identify the mechanism(s) responsible for the biosynthesis of the labeled triacylglycerols. [H-3]Arachidonate was found to be distributed in nearly equal amounts among all three glycerol positions of the triacylglycerols. In addition, analysis of intact triacylglycerols containing [H-3]arachidonate revealed that 24% of the tritium eluted from reverse-phase HPLC with triarachidonoylglycerol. Both of these findings would be expected if a significant portion of the arachidonate-containing triacylglycerols were synthesized de novo. Homogenates prepared from [H-3]arachidonate prelabeled HL-60 cells were capable of hydrolyzing the endogenous [H-3]arachidonate-containing triacylglycerols to produce mainly free fatty acids and smaller amounts of monoacylglycerols. The relatively small amount of monoacyl- and diacylglycerols produced by the lipolytic activity of the homogenates indicated that [H-3]arachidonate was hydrolyzed from all three sn-positions of the [H-3]triacylglycerols. This lipase activity had a pH optimum of 4.5 and was associated to a greater extent with the soluble fraction than in the total membrane fraction. Although it is not known whether this lipolytic activity is the same as that expressed in the intact cells, the activity of the cell-free triacylglycerol lipase was of sufficient magnitude to have easily accounted for the decrease in [H-3]triacylglycerols that was observed after transfer of the intact HL-60 cells (prelabeled with [H-3]arachidonate) to fresh media. The data suggest that transfer of arachidonate from triacylglycerols to phospholipids probably occurs through an acyltransferase utilizing a lysophospholipid and arachidonoyl-CoA.