| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The enzymes (acyl-CoA oxidase, two 3-hydroxyacyl-CoA hydratase/dehydrogenase and acyl-CoA thiolase) related to side chain degradation in bile acid biosynthesis are located in liver peroxisomes, however, the substrate specificities and/or stereospecificiies of these enzymes have not been clearly established. The aime of the present study is to clarify the stereochemical course of the side chain degradation and specificities of these enzymes.
First, we chemically synthesized 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oyl CoA, 3α, 7α, 12α-trihydroxy-5β-cholest-24-en-26-oyl CoA, 3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl CoA, and 3α, 7α, 12α-trihydroxy-24-oxo-5β-cholestanoyl CoA as the substrates for these three enzymes, respectively, and analogues of these CoA esters for the study of substrate specificities. The stereoisomers of these CoA esters were also synthesized for the study of stereospecificities.
Then, we developed the quantitative analytical method using HPLC for direct analysis of t
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he above CoA esters . The stereoisomers of C27-bile acid CoA esters were effectively separated by HPLC method. And also GC-MS method was also established for the analysis of free C27-bile acids after derivatization into methyl ester-di-methylethylsilyl ethers. These established analytical methods were highly effective for the analysis of products of enzymatic reactions and C27-bile acids in urine of patients with bile acid biosynthesis disorder.
The products analysis of the above synthesized CoA esters with related enzymes, which were prepared from rat liver peroxisomal fractions, were carried out using above established analytical method. The results are summarized as follows ;
The substrate specificities were observed in all enzymes. It was noted that the terminal methyl group is strongly recognized by 3-hydroxyacyl-CoA hydratase/dehydrogenase (DBP), and also this enzyme give (24R,25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl CoA as its hydratase activity, which is further dehydroganated its dehydrogenase activity to give 3α, 7α, 12α-trihydroxy-24-oxo-5β-cholestanoyl CoA.Another 3-hydroxyacyl-CoA hydratase/dehydrogenase (LBP) gave (24S,25S)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl CoA by its hydratse, however, LBP could not convert it to 24-oxo-compound. These results indicated that the "real e hydratse/dehydrogenase" in bile acid biosynthesis is DBP.The last degradation step by acyl-CoA thiolase was also investigated. It was found that Sterol carrier protein X has strong acyl CoA tholase activity for 24-oxo-C27-bile acids indicating the "true thiolase" in biosynthesis of bile acid. Less
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