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N-myristoyl transferase assay using phosphocellulose paper binding.
1Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Analytical Biochemistry
|December 1, 1991
Summary
A new assay for N-myristoyl-CoA:protein N-myristoyl transferase (NMT) activity was developed. This method, utilizing [3H]myristoylated peptide binding to phosphocellulose paper, identified brain tissue as having the highest NMT activity.
Area of Science:
- Biochemistry
- Enzymology
Background:
- N-myristoyl-CoA:protein N-myristoyl transferase (NMT) catalyzes the attachment of myristic acid to proteins.
- This modification is crucial for protein function and localization.
Purpose of the Study:
- To develop a novel, efficient assay for NMT activity.
- To compare NMT activity across different mammalian tissues.
Main Methods:
- A new assay was established using [3H]myristoylated peptide binding to a P81 phosphocellulose paper matrix.
- Two specific peptides, derived from cAMP-dependent protein kinase and pp60src, were used as substrates.
- Tissue extracts from rat and bovine were analyzed for NMT activity.
Main Results:
- The developed assay is rapid, reliable, and cost-effective.
- Brain tissue exhibited the highest NMT activity compared to spleen, heart, and liver in both rat and bovine.
- Myristoylation rates were linear with time and brain extract concentration under assay conditions.
Conclusions:
- The new phosphocellulose paper-based assay offers a convenient method for measuring NMT activity.
- Brain possesses the highest endogenous NMT activity among the surveyed tissues, suggesting its potential importance in neural functions.