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A radiometric assay for Ras-processing peptidase using an enzymatically radiolabeled peptide
N H Georgopapadakou1, C C Hall, T Lambros
1Department of Oncology, Roche Research Center, Nutley, New Jersey 07110-1199.
Analytical Biochemistry
|May 1, 1994
Summary
A new radiometric assay simplifies the study of Ras-processing peptidase activity. This method efficiently screens inhibitors and substrates for post-translational modification of p21ras proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ras-processing peptidase is crucial for post-translational modification of p21ras proteins.
- The Cys-aliphatic-aliphatic-any amino acid (CAAX) motif is a key target for this peptidase.
- Efficient assays are needed to study peptidase activity and identify inhibitors.
Purpose of the Study:
- To develop a simple and sensitive radiometric assay for Ras-processing peptidase.
- To enable efficient screening of substrates and inhibitors for this enzyme.
- To facilitate research into the post-translational processing of p21ras proteins.
Main Methods:
- Synthesis of a radiolabeled isoprenylated tetrapeptide substrate.
- Use of a microsomal preparation of Ras-processing peptidase from bovine liver.
- Separation of substrate and product using thin-layer chromatography.
Main Results:
- The assay demonstrated first-order kinetics with respect to the substrate.
- IC50 values approximated Km and Ki values, simplifying kinetic analysis.
- The assay does not require expensive or specialized equipment.
Conclusions:
- A simplified radiometric assay for Ras-processing peptidase has been established.
- This assay is suitable for screening potential substrates and inhibitors.
- The method aids in understanding p21ras protein post-translational processing.