Purification and properties of cytidine deaminase from escherichia coli.
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.
D001881:Borates / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D003564:Cytidine Deaminase / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D009700:Nucleoside Deaminases / Q000302:isolation & purification*; D013379:Substrate Specificity
D001881:Borates; D009700:Nucleoside Deaminases; D003564:Cytidine Deaminase