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Related Experiment Videos

A rapid and versatile method for screening endothelin converting enzyme activity

D K Little1, D M Floyd, A A Tymiak

  • 1Department of Cardiovascular Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543 4000.

Journal of Pharmacological and Toxicological Methods
|August 1, 1994
PubMed
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A new two-step assay protocol enables high-throughput screening of endothelin converting enzyme (ECE) activity. This method efficiently detects endothelin peptides using endothelial cells and optimized substrate conversion for cost-effective, sensitive assays.

Area of Science:

  • Biochemistry
  • Enzymology
  • Cell Biology

Background:

  • Endothelin converting enzyme (ECE) plays a crucial role in synthesizing vasoactive endothelin peptides.
  • Accurate and efficient assays for ECE activity are vital for drug discovery and understanding cardiovascular regulation.
  • Existing methods for ECE activity assessment can be costly and lack high-throughput capabilities.

Purpose of the Study:

  • To develop a flexible, high-throughput two-step protocol for assaying endothelin converting enzyme (ECE) activity.
  • To optimize cleavage conditions for cost-efficiency and sensitive detection of mature endothelin.
  • To characterize ECE activity in human endothelial cells using the developed assay.

Main Methods:

  • A two-step protocol involving exogenous substrate conversion and subsequent endothelin quantification.

Related Experiment Videos

  • Utilized endothelial cell monolayers and crude cell extracts as enzyme sources.
  • Employed enzyme immunoassays (EIA) and radioreceptor assays in 96-well formats for quantification.
  • Optimized cleavage conditions to minimize substrate use while maximizing sensitivity and selectivity.
  • Investigated ECE characteristics, including membrane-bound nature and sensitivity to inhibitors like phosphoramidon.

    • Main Results:

    • The developed protocol is suitable for high-throughput screening of ECE activity.
    • Optimized conditions achieved sensitive and selective detection of mature endothelin peptides.
    • Comparable endothelin formation estimates were obtained using EIA and radioreceptor assays.
    • Human umbilical vein and aorta endothelial cells preferentially convert big endothelin-1 (big ET-1).
    • ECE was identified as a membrane-bound, thiorphan-insensitive, and phosphoramidon-sensitive zinc metalloendopeptidase.
    • Similar IC50 values (around 1 microM) for phosphoramidon inhibition were observed using intact cells and membrane preparations.

    Conclusions:

    • A simple, rapid, and flexible two-step protocol for high-throughput ECE activity assays has been established.
    • The protocol enables cost-effective and sensitive detection of endothelin peptides.
    • The findings confirm ECE's role in big ET-1 conversion in human endothelial cells and its enzymatic properties.
    • This assay is well-suited for large-scale screening in drug discovery and cardiovascular research.