A novel pathway of fatty acid metabolism

大多数脂肪酸通过β-氧化途径代谢，并被用作能量来源。另一方面，一部分脂肪酸通过ω-氧化途径代谢，ω-氧化途径的第一个反应是ω-羟基化，由微粒体P450单加氧酶系统催化。本课题组从兔肝、肾和肺微粒体中分离纯化了脂肪酸羟化酶（P450），并对其底物特异性和引物结构进行了研究。为了建立γ-氧化途径，本研究从兔肝胞液中分离纯化了γ-氧化途径中的酶，并对其进行了表征：1.在NAD^+存在下，兔肝胞液能催化12-羟基十二烷酸和16-羟基十六烷酸脱氢，并能催化乙醇脱氢。产物经高效液相色谱鉴定分别为1，12-十二烷二酸和1，16-十六烷二酸 关于我们 2.醇脱氢酶具有广泛的底物特异性，也催化ω-羟基脂肪酸脱氢。但与乙醇脱氢酶活性相比，兔肝细胞中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，在兔肝胞液中存在一种独特的ω-羟基脂肪酸脱氢酶。3.从兔肝胞液中纯化了ω-羟基脂肪酸脱氢酶。羟脂肪酸脱氢酶在SDS-聚丙烯酰胺凝胶上呈单一多肽带迁移，分子量为51，000。通过BrCN降解从纯化的酶衍生的肽的氨基酸序列进行了测定。氨基酸序列与人乙醛脱氢酶有较高的同源性。4.以BrCN多肽的氨基酸序列为模板，采用PCR技术从兔肝cDNA文库中扩增出ω-羟基脂肪酸脱氢酶的cDNA。该cDNA（约500 bp）不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