Isolation of Deoxycytidine Kinase from Ehrlich Carcinoma Cells by Affinity Chromatography Based on a Substrate Analog, 2'-C-Cyano-2'-deoxy-1-β-D-arabinofuranosyl-N4-palmitoylcytosine

Hiroyuki UCHIDA; Yi-Xin CHEN; Hidetaka MORINAGA; Yuko HAYASHI; Akira MATSUDA; Tohru OBATA; Yoshio ENDO; Takuma SASAKI

doi:10.1248/bpb.22.83

Isolation of Deoxycytidine Kinase from Ehrlich Carcinoma Cells by Affinity Chromatography Based on a Substrate Analog, 2'-C-Cyano-2'-deoxy-1-β-D-arabinofuranosyl-N⁴-palmitoylcytosine

Hiroyuki UCHIDA, Yi-Xin CHEN, Hidetaka MORINAGA, Yuko HAYASHI, Akira MATSUDA, Tohru OBATA, Yoshio ENDO, Takuma SASAKI

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Keywords: deoxycytidine kinase, purification, purification, affinity chromatography, Ehrilich carcinoma cell, kinetic parameter

JOURNAL FREE ACCESS

1999 Volume 22 Issue 1 Pages 83-86

DOI https://doi.org/10.1248/bpb.22.83

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Abstract

Deoxycytidine kinase from Ehrlich carcinoma cells was purified 10400-fold by ammonium sulfate fractionation and affinity chromatography using Sepharose 4B coupled to 2'-C-cyano-2'-deoxy-1-β-D-arabinofuranosyl-N⁴-palmitoylcytosine, with a yield of 45%. The purified enzyme preparation showed a single major band with a molecular weight of 32000 on SDS-PAGE. The enzyme phosphorylated deoxyadenosine, deoxyguanosine, cytidine, and several deoxycytidine analogues as well as deoxycytidine. Also, the kinetic parameters of the enzyme for the substrates were estimated.

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