Michaelson L V, Longman A J, Sayanova O, Stobart A K, Napier J A
Institute of Arable Crops Research, Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, UK.
Biochem Soc Trans. 2002 Nov;30(Pt 6):1073-5. doi: 10.1042/bst0301073.
We have isolated a cDNA encoding the Delta(8) sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis - and trans -desaturated sphingoid bases which were not present in the wild-type yeast cells. The Delta(8) desaturated products co-eluted with known Delta(8)-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Delta(8) long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.
我们通过基于聚合酶链反应（PCR）的策略，使用针对微粒体去饱和酶保守组氨酸盒区域设计的引物，从植物普通耧斗菜中分离出了编码Δ(8)鞘脂去饱和酶的互补脱氧核糖核酸（cDNA）。该cDNA的功能通过在酿酒酵母中的表达得到证实。通过反相高效液相色谱法（HPLC）对作为其二硝基苯基衍生物的长链鞘氨醇碱进行分析，结果表明野生型酵母细胞中不存在的顺式和反式去饱和鞘氨醇碱有所积累。Δ(8)去饱和产物与已知的Δ(8) - 去饱和植物鞘氨醇共洗脱，并且这些产物的分子量通过液相色谱 - 质谱法得到确认。Δ(8)长链碱基去饱和酶也能够使二氢鞘氨醇底物去饱和。这是关于普通耧斗菜基因产物功能特性的首次报道。
