Metabolism of deoxypyrimidines and deoxypyrimidine antiviral analogs in isolated brain mitochondria.
Metabolism of deoxypyrimidines and deoxypyrimidine antiviral analogs in isolated brain mitochondria.
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DOI：
10.1111/j.1471-4159.2012.07765.x
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发表时间：
2012-07
影响因子：
4.7
通讯作者：
McKee EE
中科院分区：
文献类型：
--
作者：
McCann KA;Williams DW;McKee EE
该项目的目标是确定用于维持脑线粒体中脱氧核苷三磷酸池的脱氧嘧啶抢救途径的特征，并确定抗病毒嘧啶类似物利用或影响这些途径的程度。用标记和未标记的脱氧核苷和脱氧核苷类似物孵育大鼠脑线粒体。产品用高效液相色谱分析，联用内联式紫外光监测器和液体闪烁计数器。分离的线粒体将胸苷和脱氧胞苷运输到基质中，并很容易将它们磷酸化成一、二和三磷酸核苷酸。磷酸化的速率远远高于心脏和肝脏线粒体的磷酸化速率。脱氧尿苷的磷酸化速度比胸腺嘧啶核苷慢得多，而且只会被丢弃。AZT是一种抗病毒胸苷类似物，与胸腺嘧啶核苷被磷酸化成TMP一样容易被磷酸化成AZT-MP，但几乎没有观察到AZT-DP或AZT-TP。5.5±1.7μM的氮卓酮对胸腺嘧啶核苷的磷酸化有50%的抑制作用，但对脱氧胞苷的磷酸化无明显的抑制作用，但与分离的脑线粒体孵育时，司他夫定和拉米夫定处于惰性状态。当浓度为5.5±1.7μM时，则无抑制作用。脑线粒体的胸腺嘧啶核苷、DC和AZT的磷酸化动力学明显不同于肝脏和心脏的线粒体。
The goal of this project was to characterize deoxypyrimidine salvage pathways used to maintain deoxynucleoside triphosphate pools in isolated brain mitochondria and to determine the extent that antiviral pyrimidine analogs utilize or affect these pathways. Mitochondria from rat brains were incubated in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Products were analyzed by HPLC coupled to an inline UV monitor and liquid scintillation counter. Isolated mitochondria transported thymidine and deoxycytidine into the matrix, and readily phosphorylated both of these to mono-, di, and tri-phosphate nucleotides. Rates of phosphorylation were much higher than rates observed in mitochondria from heart and liver. Deoxyuridine was phosphorylated much more slowly than thymidine and only to dUMP. AZT, an antiviral thymidine analog, was phosphorylated to AZT-MP as readily as thymidine was phosphorylated to TMP, but little if any AZT-DP or AZT-TP was observed. AZT at 5.5 ± 1.7 μM was shown to inhibit thymidine phosphorylation by 50%, but was not observed to inhibit deoxycytidine phosphorylation except at levels > 100 μM. Stavudine and lamivudine were inert when incubated with isolated brain mitochondria. The kinetics of phosphorylation of thymidine, dC and AZT were significantly different in brain mitochondria compared to mitochondria from liver and heart.
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