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学科主题: 药学
题名:
Study on steady-state kinetics of nucleotide analogues incorporation by non-gel CE
作者: Li, Wujie1; Cao, Mou2; Pei, Lu1; Ling, Xiaomei1; Li, Bingchao2; Yang, Zhenjun2
关键词: Non-gel CE ; Nucleotide analogues ; Steady-state kinetic parameter
刊名: ELECTROPHORESIS
发表日期: 2010-02-01
DOI: 10.1002/elps.200900419
卷: 31, 期:3, 页:507-511
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology
类目[WOS]: Biochemical Research Methods ; Chemistry, Analytical
研究领域[WOS]: Biochemistry & Molecular Biology ; Chemistry
关键词[WOS]: CAPILLARY-ELECTROPHORESIS ; INTERPENETRATING NETWORK ; SEPARATION ; POLY(N,N-DIMETHYLACRYLAMIDE) ; POLYACRYLAMIDE ; POLYMERASE ; FRAGMENTS
英文摘要:

A method for studying steady-state kinetics of nucleotide analogues incorporation into DNA strand by non-gel CE (NGCE) with LIF was developed. Nucleoside analogue is a kind of antiviral agent used to inhibit viral replication in infected cells, especially HIV. Steady-state parameter K(m) for nucleotide analogues is determined to imply the relationship between nucleoside analogues and the enzyme in the DNA chain elongation and predict the antiviral efficacy in vivo. Samples were prepared by single nucleotide incorporation assays catalyzed by Taq DNA polymerase at 58 degrees C and HIV reverse transcriptase (RT) at 37 degrees C, and then were separated using NGCE under optimized conditions: 25 mmol/L Tris-boric-EDTA buffer (pH 8.0) with 7 mmol/L urea in the presence of 20%w/v PEG 35000 at 30 degrees C and -20 kV. K(m(dTTP)), K(m(d4TTP)) and K(m(AZTTP)) were measured by NGCE for the first time and their values for Taq DNA polymerase were 0.29 +/- 0.04, 32.1 +/- 3.3 and 74.5 +/- 6.6 mu mol/L, respectively. For HIV RT, the values were 0.15 +/- 0.05, 0.31 +/- 0.03 and 0.17 +/- 0.03 mu mol/L, respectively. The trend of data for HIV RT measured by NGCE was consistent with that measured by PAGE. The reported method by NGCE for the K(m) determination was powerful, sensitive and fast, and required less amounts of reagents compared with PAGE. It be employed as a reliable alternative method and further applied in other relative studies of nucleoside analogue substrates and DNA polymerases or RTs.

语种: 英语
所属项目编号: 2006AA02Z144 ; 20472006
项目资助者: National High-Tech Research and Development Program of China ; National Natural Science Foundation of China
WOS记录号: WOS:000274603200009
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/63638
Appears in Collections:北京大学药学院_化学生物学系_期刊论文

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作者单位: 1.Peking Univ, Sch Pharmaceut Sci, Dept Pharmaceut Anal, Beijing 100191, Peoples R China
2.Peking Univ, State Key Lab Nat & Biomimet Drugs, Beijing 100191, Peoples R China

Recommended Citation:
Li, Wujie,Cao, Mou,Pei, Lu,et al. Study on steady-state kinetics of nucleotide analogues incorporation by non-gel CE[J]. ELECTROPHORESIS,2010,31(3):507-511.
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