|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1996 : ¥1,700,000 (Direct Cost : ¥1,700,000)
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
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