Determination of the deoxycytidine kinase activity in cell homogenates with a non-radiochemical assay using reversed-phase high performance liquid chromatography; Identification of a novel metabolite of 2-chlorodeoxyadenosine.
A non-radioactive procedure to measure the deoxycytidine kinase (dCK) activity in crude cell free homogenates was developed. 2-Chlorodeoxyadenosine (CdA) was used as the substrate for dCK and was separated from its product 2-chlorodeoxyadenosine-5'-monophosphate (CdAMP) by reversed-phase HPLC. A complete separation of CdA and its metabolites was achieved in 30 min. The minimum amount of CdAMP that could be detected was 1 pmol. The assay was linear with reaction times up to at least 3h. With respect to the protein concentration, the reaction was linear with protein concentrations up to 760 microg/ml in the assay. An amount of 8 x 10(3) cells was already sufficient to determine the specific dCK activity in SK-N-BE(2)c cells. CdA was not only converted to CdAMP but also to 2-chloroadenine and, surprisingly, also to 2-chlorodeoxyinosine, in MOLT-3 cells. The deamination of CdA was completely inhibited by deoxycoformycin, which clearly demonstrates that CdA is a substrate for adenosine deaminase.
Bierau
Jörgen
J
Department of Clinical Chemistry, University of Amsterdam, Emma Children's Hospital, 1100 DE, The Netherlands.
Leen
René
R
Gennip
Albert H van
AH
Caron
Huib N
HN
Kuilenburg
André B P van
AB
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017338:Cladribine / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000378:metabolism*; D006801:Humans; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D017338:Cladribine; D003842:Deoxycytidine Kinase