Cytidine deaminase from Escherichia coli B. Purification and enzymatic and molecular properties

Alberto Vita, Adolfo Amici, Tiziana Cacciamani, Marina Lanciotti, Giulio Magni

Research output: Contribution to journalArticle

Abstract

Cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) from Escherichia coli has been purified to homogeneity through a rapid and efficient two-step procedure consisting of anion-exchange chromatography followed by preparative electrophoresis. The final preparation is homogeneous, as judged by a single band obtained by disc gel electrophoresis performed in the absence and presence of denaturing agents. The native protein molecular weight determined by gel filtration is 56 000. Sodium dodecyl sulfate disc gel electrophoresis experiments conducted upon previous incubation of the enzyme with dimethyl suberimidate suggest an oligomeric structure of two identical subunits of 33 000 molecular weight. The absorption spectrum of the protein reveals a maximum at 277 nm and a minimum at 255 nm. The isoelectric point is at pH 4.35. Amino acid analysis indicates an excess of acidic amino acid residues as well as six haif-cystine residues. No interchain disulfide groups have been evidenced. According to Cleland's nomenclature, kinetic analysis shows a rapid-equilibrium random Uni-Bi mechanism. Cytidine deaminase is competitively inhibited by various nucleosides. Km values for cytidine, deoxycytidine, and 5-methylcytidine are 1.8 × 10-4, 0.9 × 10-4, and 12.5 × 10-4 M, respectively.

Original languageEnglish
Pages (from-to)6020-6024
Number of pages5
JournalBiochemistry
Volume24
Issue number21
Publication statusPublished - 1985

ASJC Scopus subject areas

  • Biochemistry

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