de Vet E C, Zomer A W, Lahaut G J, van den Bosch H
Department of Biochemistry of Lipids, Centre for Biomembranes and Lipid Enzymology, Utrecht, The Netherlands.
J Biol Chem. 1997 Jan 10;272(2):798-803. doi: 10.1074/jbc.272.2.798.
Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a number of peroxisomal disorders leads to deficiencies in ether phospholipids. We have previously purified the committed enzyme for ether lipid biosynthesis, i.e. alkyl-dihydroxyacetone-phosphate synthase, to homogeneity. We have now determined the N-terminal amino acid sequence, as well as additional internal sequences obtained after cyanogen bromide cleavage of the enzyme. With primers directed against the N-terminal sequence and against a cyanogen bromide fragment sequence, a 1100-bp cDNA fragment was obtained by conventional polymerase chain reaction using first-strand cDNA from guinea pig liver as a template. The 5' and 3' ends of the cDNA were obtained by rapid amplification of cDNA ends. The open reading frame encodes a protein of 658 amino acids, containing the N-terminal amino acid sequence as well as the cyanogen bromide cleavage fragment sequences. The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids long. The latter contains a stretch of amino acids known as peroxisomal targeting signal 2. The size of the mRNA was estimated to be around 4200 nucleotides. Recombinant His-tagged alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli was enzymatically active.
过氧化物酶体是哺乳动物组织中醚脂生物合成所必需的细胞器，在许多过氧化物酶体疾病中这些细胞器的缺乏会导致醚磷脂的缺乏。我们之前已将醚脂生物合成的关键酶，即烷基二羟基丙酮磷酸合酶纯化至同质。我们现在已确定了其N端氨基酸序列以及该酶经溴化氰裂解后获得的其他内部序列。使用针对N端序列和溴化氰片段序列的引物，以豚鼠肝脏的第一链cDNA为模板，通过常规聚合酶链反应获得了一个1100bp的cDNA片段。cDNA的5'和3'末端通过cDNA末端快速扩增获得。开放阅读框编码一个由658个氨基酸组成的蛋白质，包含N端氨基酸序列以及溴化氰裂解片段序列。推导的氨基酸序列包括一个600个氨基酸长的成熟蛋白和一个58个氨基酸长的前序列。后者包含一段被称为过氧化物酶体靶向信号2的氨基酸序列。mRNA的大小估计约为4200个核苷酸。在大肠杆菌中表达的重组His标签烷基二羟基丙酮磷酸合酶具有酶活性。
