Document Detail


High-throughput assay for long chain fatty acyl-CoA elongase using homogeneous scintillation proximity format.
MedLine Citation:
PMID:  19505229     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Elongase of very-long-chain fatty acid (Elovl) 6 is a rate-limiting enzyme that is responsible for the elongation of long-chain fatty acids such as palmitoic acid (C16). Elovl6 is abundantly expressed in liver and adipose tissue, and the expression levels in these tissues are up-regulated in obese animals. Furthermore, Elovl6-deficient mice display improved glucose homeostasis and insulin sensitivity, suggesting that Elovl6 might be a potential therapeutic target for metabolic disorders. From the drug discovery point of view, it is critical to establish a high-throughput screening (HTS) assay for the identification of therapeutic agents. Conventional assay methods for fatty acid elongases include an extraction step for respective radioactive products from the reaction mixtures, which is labor-intensive and not feasible for HTS. In this study, we utilized the acyl-coenzyme A (CoA) binding protein (ACBP) as a molecular probe to detect radioactive long-chain acyl-CoA, a direct product of Elovl6. Recombinant ACBP binds stearoyl-CoA but not malonyl-CoA, enabling specific detection of the radioactive product in the homogenous reaction mixture without the liquid extraction step. Finally, combination of ACBP and scintillation proximity assay beads led to specific detection of Elovl6 activity with appropriate window and reproducibility amenable to HTS (signal-to-background noise ratio of approximately 13.0-fold, Z' = 0.85). The assay system described here has the potential to enable identification of small compounds that modify fatty acid elongase activity and assessment of the therapeutic potential of acyl-CoA elongases.
Authors:
Ken Shimamura; Yasuhisa Miyamoto; Hidefumi Kitazawa; Tsutomu Kobayashi; Hidehito Kotani; Shigeru Tokita
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Assay and drug development technologies     Volume:  7     ISSN:  1540-658X     ISO Abbreviation:  Assay Drug Dev Technol     Publication Date:  2009 Apr 
Date Detail:
Created Date:  2009-06-09     Completed Date:  2009-08-11     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101151468     Medline TA:  Assay Drug Dev Technol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  124-32     Citation Subset:  IM    
Affiliation:
Tsukuba Research Institute, Banyu Pharmaceutical Company, Ltd., Ibaraki, Japan.
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MeSH Terms
Descriptor/Qualifier:
Acetyltransferases / metabolism*
Acyl Coenzyme A / metabolism
Diazepam Binding Inhibitor / metabolism
Drug Discovery
Humans
Scintillation Counting
Chemical
Reg. No./Substance:
0/Acyl Coenzyme A; 0/Diazepam Binding Inhibitor; 362-66-3/stearoyl-coenzyme A; EC 2.3.1.-/Acetyltransferases; EC 2.3.1.-/fatty acid elongases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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