A novel pathway of fatty acid metabolism

几乎所有脂肪酸都是通过β -氧化途径代谢并作为能量来源利用的。另一方面，一部分脂肪酸通过w-氧化途径代谢，w-氧化途径的第一个反应是w-羟基化，由微粒体P450单糖酶系统催化。本课课组从兔肝脏、肾脏和肺微粒体中纯化出脂肪酸w-羟化酶（P450），并对其底物特异性和一级结构进行了表征。为了建立w-氧化途径，本研究从兔肝细胞质中纯化了组成该途径的酶，并对其进行了表征。兔肝细胞质溶胶在NAD^+存在下可催化12-羟基十二烷酸和16-羟基十六烷酸脱氢，也可催化乙醇脱氢。采用高效液相色谱、气相色谱-质谱联用技术鉴定产物分别为1,12-十二烷二酸和1,16-十六烷二酸。醇脱氢酶具有广泛的底物特异性，也能催化w-羟基脂肪酸的脱氢。但与乙醇脱氢酶活性相比，兔肝细胞醇中w-羟基脂肪酸脱氢酶活性的热稳定性不稳定。这一结果表明，兔肝细胞质中存在一种独特的w-羟基脂肪酸脱氢酶。从兔肝细胞质中纯化得到w-羟基脂肪酸脱氢酶。w-羟基脂肪酸脱氢酶在sds -聚丙烯酰胺凝胶上以分子量为51,000的单多肽带迁移。测定了BrCN降解纯化酶得到的肽的氨基酸序列。氨基酸序列与人醛脱氢酶具有较高的同源性。从兔肝脏cDNA文库中，利用BrCN肽的氨基酸序列推导出w-羟基脂肪酸脱氢酶的寡核苷酸序列，用PCR方法扩增出w-羟基脂肪酸脱氢酶cDNA。该cDNA（约500bp）未包含开放阅读框的全长。我们从兔肝cDNA文库中克隆了全长cDNA。少

Almost of fatty acids are metabolized viabeta-oxidation pathway and utilized as energy source. On the other hand, a part of fatty acids are metabolized via w-oxidation pathway, The first reaction of w-oxidation pathway is w-hydroxylation which catalized by microsomal P450 monoowygenase system. Fatty acid w-hydroxylases (P450) had been purified from rabbit liver, kidney and lung microsomes, and their substrate specificities and primery structures had been characterized well by our research group. To establish w-oxidation pathway, the enzymes composed of this pathway were purified from rabbits hepatic cytosol and characterized in this research.1.Cytosol from rabbit liver was found to catalyze the dehydrogenation of 12-hydroxy dodecanoic acid and 16-hydroxy hexadecanoic acid in the presence of NAD^+ as well as the dehydrogenation of ethanol. The products were identified as 1,12-dodecane dioic acid and 1,16-hexadecane dioic acid, respectively, by using high performance liquid chromatograph … More y and gas chromatography-mass spectrometry.2.Alcohol dehydrogenase, which has a broad substrate specificity, also catalyze dehydrogenation of w-hydroxy fatty acid. But thermal stability of the w-hydroxy fatty acid dehydrogenase activity in rabbit hepatic cytosl was unstable in compare with ethanol dehydrogenase activity. This result indicates the presence of a unique dehydrogenase specific for w-hydroxy fatty acids in rabbit hepatic cytosol.3.w-hydroxy fatty acid dehydrogenase was purified from rabbit hepatic cytosol. w-Hydroxy fatty acid dehydrogenase migrated as single polypeptide band with molecular weight of 51,000 on SDS-polyacryl-amide gel. Amino acid sequences of the peptides derived from purified enzyme by BrCN degradation were determined. The amino acid sequences showed high homology with human aldehyde dehydrogenase.4.cDNA of w-hydroxy fatty acid dehydrogenase was amplified from rabbit hepatic cDNA library by PCR using oligonucleotides which sequences were deduced from the amino acid sequences of BrCN peptides. This cDNA (about 500bp) did not containe full length of the open reading frame. We have been cloning of cDNA contained full length from rabbit hepatic cDNA library. Less