Isoenzymatic forms of human cytidine deaminase

Abstract

Cytidine deaminase (CDA) purified from human placenta revealed the presence of five isoenzymatic forms that differ only in their isoelectric point. Since human cytidine deaminase exists in two variants (CDA 1 and CDA 2) with a non-conservative amino acid substitution at codon 27, in this work we demonstrate that these two variants may combine together in vitro, giving five CDA isoforms as observed in vivo from human placenta. For this purpose, each of the two forms of CDA was purified close to homogeneity and dissociated into monomers in the presence of a small amount of sodium dodecyl sulfate as a dissociating agent. The monomers were mixed together and subjected to anion-exchange chromatography and to chromatofocusing analysis in order to visualize the formation of the five isoforms. Furthermore, for both CDA 1 and CDA 2 some substrates and inhibitors of CDA were assayed, with the aim of demonstrating different kinetic behavior between the two natural variants.

Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase

“…Therefore, the relative order of catalytic activities among the three CDA variants for Ara-C (WT = CDA27Gln . CDA70Thr) was similar to previous studies (Yue et al, 2003;Vincenzetti et al, 2004).…”

Creation of zebularine-resistant human cytidine deaminase mutants to enhance the chemoprotection of hematopoietic stem cells

“…The kinetic constants of the wild-type enzyme were very similar to those previously reported, while the three mutants displayed diverse kinetic characteristics (Laliberte et al, 1992;Vincenzetti et al, 1996Vincenzetti et al, , 2004. Comparing the results of wild-type hCDA2 toward CdR, all three mutants displayed a significant overall reduction in catalytic efficiency (k cat /K M ) ranging from 6.5-to 10-fold lower.…”

“…Growth curve experiments with CdR in the presence or absence of zebularine served to further validate the results of plate assays. Both human and bacterial CDA enzymes have been the focus of numerous alignment and site-directed mutagenesis studies as a means to elucidate conserved residues, those that participate in substrate interactions and catalytic behavior (Betts et al, 1994;Cambi et al, 1998;Johansson et al, 2002;Vincenzetti et al, 2004Vincenzetti et al, , 2008Vincenzetti et al, , 2013Chung et al, 2005;Costanzi et al, 2006;Teh et al, 2006). Human CDA exists as a homotetramer with each of the subunits coordinating a zinc ion through three cysteine residues (C65, C99 and C102) (Cambi et al, 1998).…”

“…E. coli strain SØ5201 (MC1061cdd:: Tn10 pyr::Kan), the pyrimidine requiring CDA-negative derivative of MC1061, was kindly provided by Dr Alberto Vita (Vincenzetti et al, 1996(Vincenzetti et al, , 2004. E. coli SØ5201 was lysogenized with λDE3 according to the manufacturer's instructions (Novagen, Madison, WI, USA).…”

“…There are two known natural variants of hCDA, hCDA1 and hCDA2, which differ by a single amino acid at position 27: glutamine (Q) in hCDA1 and lysine (K) in hCDA2 (Kuhn et al, 1993;Laliberte and Momparler, 1994;Kirch et al, 1998;Vincenzetti et al, 2004). Studies have reported that hCDA2 exerts a 1.3-to 3.3-fold higher deamination rate toward AraC Fig.…”