PMID:6386817
| Citation |
Ashley, GW and Bartlett, PA (1984) Purification and properties of cytidine deaminase from escherichia coli. J. Biol. Chem. 259:13615-20 |
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| Abstract |
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. |
| Links | |
| Keywords |
Borates/pharmacology; Chromatography, Affinity/methods; Cytidine Deaminase/isolation & purification; Cytidine Deaminase/metabolism; Escherichia coli/enzymology; Kinetics; Molecular Weight; Nucleoside Deaminases/isolation & purification; Substrate Specificity |
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