碩士 / 中山醫學大學 / 營養學研究所 / 93 / In this study, the srarch tool BLAST(Basic Local Alignment Search Tool), based on NCBI（National Center for Biotechnology Information）was used against nucleotide and protein database. The analysis found a Dimeric Dihydrodiol Dehydrogenase（dimeric DD）of zebrafish（NCBI accession number BI326319）and human（NCBI accession number NM_014475.2/NP_055290）.The dimeric DD cDNA of zebrafish and human were successfully amplified with a continuous open reading frame of 1002 and 1005 bps encoding a protein of 299 and 334 amino acid with a calculated molecular mass of 36.63 and 36.74 kDa, respectively.
Zebrafish and human dimeric DD were cloned and expressed with a 6-Histidine tag for specific purification. The purified recombinant protein have mobility of 36.63 kDa and 36.74 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Northern Blotting analysis showed that dimeric DD is expressed in the rat tissues, such as brain, heart, lung, liver, spleen, intestine, kidney, muscle, and testis. Following expression, purification, and the kinetic studies, using Maltose、D-Glucose、D-Mannose、D-Xylose、D-Fructose、D-Arabinose、D-Ribose、D-Galactose and NAD+ as substrates, the specific activities of Ni2+ column purified recombinant zebrafish dimeric DD were 186、170、146、140、136、54、41 unit/mg, respectively. Therefore, this enzyme may belong to the novel Dimeric Dihydrodiol Dehydrogenase, which uses carbohydrate as substrates.
| Identifer | oai:union.ndltd.org:TW/093CSMU5513022 |
| Date | January 2005 |
| Creators | Chien-Ju, 馬千茹 |
| Contributors | 劉德中 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 71 |
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