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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
Summary
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.
- 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.