大鼠脑线粒体生物大分子合成过程中能量需求的研究

Studies on energy demand of biological large molecules syntheses in mitochondria isolated from rat brain in vitro

目的 探讨线粒体内RNA和蛋白质合成过程中的能量需求及其ATP的调节作用。方法 组织匀浆 梯度离心法分离并纯化大鼠脑皮质线粒体 ,氧电极法鉴定线粒体氧化呼吸及其偶联活性 ;体外无细胞 (Cell freeinvitro)线粒体3H UTP和3H Leucine掺入法分别测定细胞器的转录与翻译活性。结果 线粒体RNA和蛋白质合成中所需的ATP可来自线粒体自身氧化产生 ,外源ATP的加入可使转录活性增加 3 .7倍 ,而翻译活性仅提高 1 7% ,在既无外源ATP加入又无内源性产生时 ,线粒体蛋白翻译活性仍为最大活性的 5 3 1 % ;ATP浓度对RNA和蛋白质合成的影响呈双相性 ,1mmol/L时转录以及翻译活性最高 ,高浓度或低浓度均可产生浓度依赖性抑制效应。结论 ATP可在转录与翻译两个水平影响线粒体RNA和蛋白质合成 ,ATP对mtDNA表达的调节是一种经济、积极有效的反馈调节方式。

Objective To investigate the energy demand and its regulating role of ATP in RNA and protein syntheses in mitochondria. Methods Mitochondria were isolated from rat cerebral cortex and purified with a homogenizing and centrifuging program. Mitochondrial respiration and coupled activity were determined with oxygen electrode methods. The transcriptional and translational activities of organelles were measured with 3 H UTP and 3 H Leucine incorporation in cell free mitochondria system in vitro . Results ATP required in the process of mitochondrial RNA and protein synthesis came from either endogenous generation by mitochondria per se or exogenous addition. Exogenous ATP addition made a 3.7 fold increase in transcription but only 17% increase in translation. A 53.1% of maximum activity in mitochondrial translation still remained under neither exogenous addition nor endogenous generation of ATP. Furthermore, the ATP concentration in the medium showed biphasic pattern on synthesis of RNA and protein in organelles. The maximum activity in mitochondrial transcription or translation in vitro was observed when ATP concentration was 1 mmol/L in incubation medium. There were ATP concentration dependent inhibitions in transcription and translation with ATP concentration above or under 1 mmol/L in the medium. Conclusion ATP can effect mitochondrial RNA and protein synthesis on both transcription and translation levels. The regulation of ATP in mtDNA expression was an economic, active and effective feedback regulating way.