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首页> 外文期刊>Biochemistry >Role of the Hydrophobic Segment of Diacylglycerol Kinase epsilon
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Role of the Hydrophobic Segment of Diacylglycerol Kinase epsilon

机译：二酰基甘油激酶ε疏水部分的作用

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摘要

Diacylglycerol kinase epsilon (DGK(DELTA)_(epsilon)) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids. We have evaluated the contributions of this segment to the membrane interactions and functions of this protein. To test the role of the hydrophobic segment, we have compared the properties of DGK epsilon with those of a truncated form of the protein (DGK(DELTA)_(epsilon)) lacking the 40 N-terminal amino acids, which includes the hydrophobic segment. The proteins were expressed in COS-7 cells from a gene for human DGK epsilon or from a gene for a truncated form (DGK(DELTA)_(epsilon)), both of which had a FLAG tag at the amino terminus. Full-length FLAG-DGK epsilon and truncated FLAG-DGK(DELTA)_(epsilon) were both more specific for 1-stearoyl-2-arachidonoyl-sn-glycerol than for 1,2-dioleoyl-sn-grycerol. 1-Stearoyl-2-linoleoyl-sn-glycerol exhibited intermediate specificity for both forms of the enzyme. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increases the catalytic rate constant for all three substrates and may suggest a role in the negative regulation of this enzyme. A full-length DGK epsilon with a C-terminal His tag exhibited substrate specificity similar to that of the other two forms of the enzyme, indicating that the nature and position of the epitope tag did not strongly affect this property. Using an ultracentrifugation floatation assay, we showed that at neutral pH DGK(DELTA)_(epsilon) is extracted with 1.5 M KCl while DGK epsilon remains essentially fully membrane bound. The full-length protein had a weak tendency to oligomerize in the presence of weak detergents. DGK epsilon was monomeric on SDS-PAGE but exhibited partial dimerization with low concentrations of perfluorooctanoic acid. The major conclusions of this work are that the hydrophobic domain of DGK epsilon does not contribute to substrate specificity but plays a role in permanently sequestering the enzyme to a membrane.
机译：二酰基甘油激酶ε（DGKΔ_ε）在哺乳动物DGK同工型中是独特的，具有一段疏水性氨基酸。我们已经评估了这一部分对这种蛋白质的膜相互作用和功能的贡献。为了测试疏水性片段的作用，我们将DGK epsilon的特性与截短形式的蛋白质（DGKΔ_ε）的特性进行了比较，该蛋白缺乏40个N端氨基酸，其中包括疏水性片段。这些蛋白质从人DGKε基因或截短形式的基因（DGKδ_ε）在COS-7细胞中表达，两者均在氨基末端带有FLAG标签。全长FLAG-DGKε和截短的FLAG-DGKδ_ε都比1-1,2-油酰基-sn-甘油对1-硬脂酰基-2-花生四烯酰基-sn-甘油的特异性更高。 1-硬脂酰基-2-亚油酰基-sn-甘油对两种形式的酶均表现出中等特异性。结果表明，酶的截短形式保持了对在sn-2位上存在花生四烯酰基的脂质的底物特异性。截短增加了所有三种底物的催化速率常数，并可能暗示了该酶的负调控作用。具有C末端His标签的全长DGKε显示出与酶其他两种形式相似的底物特异性，表明表位标签的性质和位置并未强烈影响该特性。使用超速离心浮选测定法，我们显示了在中性pH下，用1.5M KCl提取DGKΔ_ε，而DGKε基本上保持完全膜结合。在弱去污剂的存在下，全长蛋白质的寡聚趋势很弱。 DGKε在SDS-PAGE上为单体，但在低浓度的全氟辛酸下表现出部分二聚作用。这项工作的主要结论是，DGKε的疏水结构域不有助于底物特异性，但在将酶永久螯合到膜中起作用。

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