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In vitro Methylation Assay to Study Protein Arginine Methylation
Published on: October 5, 2014
Expression, purification, and characterization of human type II arginase
D M Colleluori1, S M Morris, D E Ash
1Temple University School of Medicine, Department of Biochemistry, Philadelphia, Pennsylvania 19140, USA.
Archives of Biochemistry and Biophysics
|May 24, 2001
Summary
Human type II arginase, an enzyme involved in amino acid metabolism, shows distinct properties from type I. Its unique characteristics may lead to new treatments for smooth muscle disorders like erectile dysfunction.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Human type II arginase is a mitochondrial enzyme distinct from type I, primarily involved in ornithine production rather than urea excretion.
- Type II arginase plays a role in regulating nitric oxide biosynthesis by influencing arginine availability.
- Understanding the biochemical properties of type II arginase is crucial for developing targeted therapies.
Purpose of the Study:
- To characterize recombinant human type II arginase.
- To investigate the kinetic properties and inhibition patterns of type II arginase.
- To explore the potential for isozyme-selective inhibitors.
Main Methods:
- Expression and purification of recombinant human type II arginase in E. coli.
- Enzyme kinetics assays to determine Km for arginine.
- Inhibition studies using borate and ornithine.
Main Results:
- Recombinant human type II arginase was purified and characterized.
- The Michaelis constant (Km) for arginine was determined to be approximately 4.8 mM at physiological pH.
- Type II arginase exists mainly as a trimer; borate is a noncompetitive inhibitor, and ornithine is a weak inhibitor.
Conclusions:
- Significant isozyme-selectivity exists between type I and type II arginases regarding substrate, product, and inhibitor binding.
- These findings support the development of selective type II arginase inhibitors.
- Selective inhibitors could offer therapeutic potential for smooth muscle disorders, including erectile dysfunction.
