| Project/Area Number |
60570123
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | National Cardiovascular Center |
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Principal Investigator |
TANABE Tadashi National Cardiovascular Center Research Institute, Laboratory for Experimental Technology, Laboratory Chief, その他, 研究員 (60025624)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1986: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1985: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Fatty Acid Biosynthesis / Regulation Mechanism / Acetyl-CoA Carboxylase / Degradation / 1次構造 |
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| Research Abstract |
Abnormal lipid metabolism, such as hyperlipidemia and obesity, causes cardiovascular diseases. For the prevention and therapy of the diseases we have been studied the regulation mechanism of fatty acid biosynthesis. This is regulated at the carboxylation step of acetyl-CoA to form malonyl-CoA, that is catalyzed by acetyl-CoA carboxylase(ACC). The activity of the enzyme is controlled both by change in the quantity of ACC and by change in the catalytic efficiency of the enzyme molecule. In order to make clear the former regulation mechanism the degradation of ACC was studied in the project. Furthermore, the primary structure of ACC was investigated for the elucidation of the latter control mechanism.
1. Purified chicken liver ACC was subjected to the proteolysis with the fractionated chicken liver lysosomal extracts by phosphocellulose column chromatography. It was found that cathepsin L is the major lysosomal proteinase at least in the initial phase of the degradation of ACC. Thus, cathe
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psin L was purified to homogeniety from chicken liver and characterized. This firstly isolated avian cathepsin L has quite similar prperties to those of mammalian enzymes. Amino acid sequence analysis of the enzyme clearly demonstrated that cathepsin L belongs to the papain super family. Cathepsin L cleaves at the midpoint of the polypeptide chain.
2. ACC is a biotin enzyme with a highly-integrated protein structure. Animal ACC exhibits a Mr of 220-260 kDa and its cellular content is low. Therefore, we have attempted to deduce the primary structure of ACC from the nucleotide sequence of the cloned ACC cDNA. The cDNA library of the chicken liver poly(A) RNA was constructed according to Okayama and Berg. The library was screened with oligonucleotide probes synthesized on the basis of the partial amino acid sequences. After screening 6 x <10^6> trasformants a plasmid containing 3.8 kb ACC cDNA. The amino acid sequence deduced from the nucleotide sequence contained the biotin binding site of Met-Lys-Met. Further study to determine the complete amino acids sequence of ACC is in progress. Less
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