Cytidine deaminase isolation

An example of a successful application of affinity chromatography is the isolation of the enzyme cytidine deaminase from cells of E. coli. Cytidine was linked covalently via long spacer arms to the agarose beads as in the following diagram ... 

Cytarabine (ara-C) is an arabinose analog of cytosine. Cytarabine was originally isolated from sponges, but is now produced synthetically. Ara-C is phosphorylated to its active triphosphate form (ara-CTP) within tumor cells. Ara-CTP inhibits DNA polymerase, an enzyme responsible for strand elongation. It is also incorporated directly into DNA, where it inhibits the replication of DNA and acts as a chain terminator to prevent DNA elongation. Activation of ara-C is opposed by deaminase enzymes, particularly cytidine deaminase, which degrades ara-C to an inactive form, ara-U. " " ... 

Muto T, Muramatsu M, Taniwaki M, Kinoshita K, Honjo T. Isolation, tissue distribution, and chromosomal localization of the human activation-induced cytidine deaminase (AID) gene. Genomics. 2000 68 85-8. 

Using this approach, 20 kg of optically pure Epivir was isolated to support the development program. For further scale-up. the process was transferred to our production site at Ulverston, Cumbria, where several tons of Epivir were produced using immobilized cytidine deaminase from the recombinant strain. 

The chart in Fig. 2 shows an alternate path for the formation of dUMP by direct deamination of dCMP. This may be how cytidine could be converted to thymidylate in the cases cited above [125,126]. However, this deaminase is not usually detected in E. coli but is induced by infection with T(even) phages [132,133]. It has also been purified from chick embryo and mammalian tissues, and its properties have been extensively analyzed [134-136]. It acts as a typical allosteric enzyme in both the phage-infected E. coli and animal systems. Homotropic substrate interaction is evident, and this is modified by dCTP as an activator, and by dTTP (sometimes dGMP) as an allosteric inhibitor. This type of control apparently functions to regulate the level of dTTP by feedback inhibition and by activation when the supply of dTTP is depleted. Cytidine deaminase (EC 3.5.4.5) isolated from sheep liver [137] appears to have the same allosteric properties, with the same positive and negative effectors, as those of dCMP deaminase. The latter enzyme is also induced by phage infection in B. subtiUs, and in contrast to the deaminase from all other sources it does not show allosteric inhibition or activation by any nucleotide [138]. 

Candida utilis is grown to high biomass concentrations and the extracted RNA is subsequently hydrolysed into the four 5 nucleotides adenosine 5 -monophosphate (AMP), GMP, cytidine and uridine 5 -monophosphate by crude nuclease PI from Penicillium the desired nucleotides are isolated by ion-exchange chromatography and AMP is converted to IMP by adenyl deaminase from Aspergillus [22, 36]. 

Deoxycytidylate deaminase was first isolated from sea urchin e s by Scarano and has since been demonstrated in many animal tissues the deaminases from spleen and chick embryo have been partly purified (23, 24). The enzyme requires that the substrate must be a 4-aminopyrimidine deoxyribonucleoside 5 -monophosphate, and will accept the following substituents at the pyrimidine 5-position methyl, hydroxymethyl, fluoro, iodo, and bromo. Notably, the following are not deaminated cytidine, deoxycytidine, eytidylate, dCDP, and dCTP. The concentration of this enzyme in cells appears to be related to their proliferative state in that the enzyme is found in growing tissues, but activities are low in adult tissues such as liver. 

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