Purification and properties of cytidine deaminase from escherichia coli
| Autor: | Paul A. Bartlett, G W Ashley |
|---|---|
| Rok vydání: | 1984 |
| Předmět: |
chemistry.chemical_classification
Gel electrophoresis Chromatography Chemistry Sodium Size-exclusion chromatography chemistry.chemical_element Cytidine Cell Biology Cytidine deaminase medicine.disease_cause Biochemistry chemistry.chemical_compound Enzyme Thiol medicine Molecular Biology Escherichia coli |
| Zdroj: | Journal of Biological Chemistry. 259:13615-13620 |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/s0021-9258(18)90738-6 |
| Popis: | A convenient and efficient procedure for the purification of cytidine deaminase (EC 3.5.4.5) from Escherichia coli is reported. The key step involves adsorption of the enzyme from a crude ammonium sulfate fraction onto a cytidine-containing affinity resin, followed by elution with 0.5 M borate buffer. Subsequent chromatography on DEAE-Sepharose results in an overall 1690-fold purification, yielding enzyme with a specific activity of 118 units/mg. Cytidine deaminase has an apparent molecular weight of 54,000 as determined by gel filtration, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a band at molecular weight 35,000. Cytidine deaminase is inhibited by 5-(chloromercuri)cytidine with kinetic behavior typical of active-site-directed inactivation, with KD = 0.09 mM and kinact = 1.25 min-1. The enzyme is protected against inactivation in the presence of substrate, and the inhibition is reversed with high concentrations of mercaptoethanol. This suggests that inactivation is the result of a mercaptide formation between the mercury and an active-site thiol. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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