Human ras-converting enzyme (hRCE1) endoproteolytic activity on K-ras-derived peptides
I Hollander1, E Frommer, R Mallon
1Oncology Research, Wyeth-Ayerst Research, 401 North Middletown Road, Pearl River, New York, 10965, USA.
Analytical Biochemistry
|October 20, 2000
Summary
Researchers developed a direct fluorescent assay to study hRCE1 protease activity, crucial for Ras protein modification. This assay effectively monitors enzyme function and aids in discovering new inhibitors for prenyl protein proteases.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- hRCE1 is a human endoprotease involved in Ras post-translational modification and membrane localization.
- Ras proteins require proper modification for their function in cell signaling.
Purpose of the Study:
- To characterize the activity and substrate specificity of the hRCE1 protease.
- To develop a direct fluorescent assay for monitoring hRCE1 proteolytic activity.
Main Methods:
- Utilized farnesylated 9 amino acid peptides, including KSKTKC(farnesyl)VIM and modified versions with fluorescent labels (MCA, ABZ) and quenchers.
- Employed High-Performance Liquid Chromatography (HPLC) for initial product analysis.
- Established a 96-well microtiter plate-based fluorescent assay for direct activity monitoring.
Main Results:
- hRCE1 specifically cleaves KSKTKC(f)VIM peptides between the C(f) and V positions.
- The peptide sequence KSKTKC(f)VI demonstrated to be a better substrate than KSKTKC(f)VIM.
- The developed fluorescent assay successfully detected hRCE1 activity and was validated by its sensitivity to the inhibitor RPI.
Conclusions:
- A direct fluorescent microtiter plate assay effectively monitors hRCE1 proteolytic activity.
- This assay is a valuable tool for further enzyme characterization and the discovery of novel hRCE1 inhibitors.
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