拟南芥新型磷脂酶A1 cDNA在酵母中的表达。

Alexandre Noiriel, Pierre Benveniste, Antoni Banas, Sten Stymne, Pierrette Bouvier-Navé
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引用次数: 39

摘要

在寻找编码植物甾醇酰基转移酶的cdna过程中,我们从拟南芥中分离出4个全长cdna,它们编码与动物卵磷脂基本相同的蛋白质:胆固醇酰基转移酶(LCATs)。在各种酵母菌株中表达其中一种cdna, atcat3 (At3g03310),导致三酰甘油含量增加一倍。此外,对转化的野生型酵母进行全脂质分析表明,其磷脂含量低于空白质粒转化的酵母,而溶血磷脂和游离脂肪酸含量增加。当来自atlcat3转化酵母的微体与二-[1-14C]油基磷脂酰胆碱孵育时,溶血磷脂和游离脂肪酸部分都被高度标记,而与来自对照酵母的微体孵育时,这些部分的标记可以忽略不计。此外,当将转化为AtLCAT3的酵母微体与sn-1-或sn-2-[1-14C]酰基磷脂酰胆碱孵育时,游离脂肪酸和溶血磷脂酰胆碱组分之间的标记分布强烈表明AtLCAT3具有磷脂酶A1活性。通过对1-肉豆蔻酰基,2-油基磷脂酰胆碱水解的气相色谱分析证实了该磷脂酶的sn-1特异性。磷脂酰乙醇胺和磷脂酸也可被AtLCAT3水解,但效率低于磷脂酰胆碱。溶血磷脂酰胆碱是弱底物,而三棕榈酰甘油和油酸胆固醇则完全不被水解。这种新的拟蓝藻磷脂酶A1在pH 6-6.5和60-65℃时表现出最佳活性,并且似乎不受Ca2+的影响。它的序列与所有其他已知的磷脂酶无关。进一步的研究正在进行中,以阐明其生理作用。
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Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.

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