Spectroscopic study of fluorescent peptides for prenyl transferase assays
Spectroscopic study of fluorescent peptides for prenyl transferase assays
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DOI：
10.1016/j.jpba.2004.11.006
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发表时间：
2005-03-09
影响因子：
3.4
通讯作者：
Goossens, JF
中科院分区：
文献类型：
--
作者：
Goossens, L;Deweer, S;Goossens, JF
以大鼠脑细胞质组分为酶源，用荧光法研究了戊烯基转移酶反应。合成了4个与Ras同工型c端序列对应的丹化肽。考察了不同洗涤剂对四种多肽的法尼基化或香叶基化的影响。研究了十二烷基麦芽糖苷（一种非离子型洗涤剂）对法尼基转移酶或香叶基转移酶活性的剂量依赖性影响。此外，研究了温度的影响，并利用这些方法测定了底物的Michaelis-Menten常数（K-m）： danyl - gcvls （1.8 μ M）、danyl - gcvvm （3.2 μ M）、danyl - cvim （3.4 μ M）、danyl - gcvll （8.4 μ M）和FPP （22.6 μ M）对FTase活性的影响。以GGPP为共底物，测定了丹西- gcvls、丹西- gcvvm和丹西- cvim三种底物的GGTase活性，其K-m值均大于50 μ M，而丹西- gcvll序列和GGPP共底物的K-m值分别为7.6和5.4 μ M。选择性转移酶抑制剂B-581、FTI 276和GGTI 287的IC50值分别为34、0.8和18 nM，以丹酰- gcvls为底物（抑制FTase）。当以丹酰gcvll作为底物（GGTase抑制）时，B-581、FTI 276和GGTI 287的IC50值分别为5100、75和5 nM。然后，这种开发的方法可以评估所有三种抑制剂的选择性。(c) 2004 Elsevier B.V.版权所有
A study of the prenyl transferase reactions was performed by fluorescence using rat brain cytosol fractions as an enzyme source. Four dansylated peptides corresponding to the C-terminal sequence of Ras isoforms were synthesised. The effects of different detergents on the farnesylation or geranylgeranylation of the four peptides were evaluated. Dose-dependent effects of dodecyl-maltoside, a non-ionic detergent, on the farnesyl transferase or geranylgeranyl transferase activities were observed with all peptide substrates. Additionally, the effect of temperature was investigated and these assays were applied to determine Michaelis-Menten constants (K-m) of the substrates: dansyl-GCVLS (1.8 mu M), dansyl-GCVVM (3.2 mu M), dansyl-CVIM (3.4 mu M) and dansyl-GCVLL (8.4 mu M) and FPP (22.6 mu M) for FTase activity. Using GGPP as co-substrate, GGTase activity was measured with K-m values superior to 50 mu M for all the three substrate dansyl-GCVLS, dansyl-GCVVM, or dansyl-CVIM, whereas values of 7.6 and 5.4 mu M were calculated for the dansyl-GCVLL sequence and GGPP co-substrate, respectively.IC50 values of selective pretty transferase inhibitors, B-581, FTI 276 and GGTI 287 have been measured to 34, 0.8 and 18 nM, respectively, using dansyl-GCVLS as substrate (FTase inhibition). When dansyl-GCVLL is used as substrate (GGTase inhibition) the IC50 values are 5100, 75 and 5 nM for B-581, FTI 276 and GGTI 287, respectively. Then, this developed method allowed to evaluate the selectivity of all the three inhibitors tested. (c) 2004 Elsevier B.V. All rights reserved.