Dejean L, Beauvoit B, Bunoust O, Fleury C, Guérin B, Rigoulet M
Institut de Biochimie et Génétique Cellulaires du CNRS, Université Bordeaux 2, France.
Biochim Biophys Acta. 2001 Jan 19;1503(3):329-40. doi: 10.1016/s0005-2728(00)00210-3.
Although on-line calorimetry has been widely used to detect transitions in global metabolic activity during the growth of microorganisms, the relationships between oxygen consumption flux and heat production are poorly documented. In this work, we developed a respirometric and calorimetric approach to determine the enthalpy efficiency of respiration-linked energy transformation of isolated yeast mitochondria and yeast cells under growing and resting conditions. On isolated mitochondria, the analysis of different phosphorylating and non-phosphorylating steady states clearly showed that the simultaneous measurements of heat production and oxygen consumption rates can lead to the determination of both the enthalpy efficiency and the ATP/O yield of oxidative phosphorylation. However, these determinations were made possible only when the net enthalpy change associated with the phosphorylating system was different from zero. On whole yeast cells, it is shown that the simultaneous steady state measurements of the heat production and oxygen consumption rates allow the enthalpy growth efficiency (i.e. the amount of energy conserved as biomass compared to the energy utilised for complete catabolism plus anabolism) to be assessed. This method is based on the comparison between the calorimetric-respirometric ratio (CR ratio) determined under growth versus resting conditions during a purely aerobic metabolism. Therefore, in contrast to the enthalpy balance approach, this method does not rely on the exhaustive and tedious determinations of the metabolites and elemental composition of biomass. Thus, experiments can be performed in the presence of non-limiting amounts of carbon substrate, an approach which has been successfully applied to slow growing cells such as yeast cells expressing wild-type or a mutant rat uncoupling protein-1.
尽管在线量热法已被广泛用于检测微生物生长过程中整体代谢活性的转变，但氧消耗通量与热量产生之间的关系却鲜有文献记载。在这项工作中，我们开发了一种呼吸测定和量热测定方法，以确定在生长和静止条件下分离的酵母线粒体和酵母细胞呼吸相关能量转化的焓效率。对于分离的线粒体，对不同的磷酸化和非磷酸化稳态的分析清楚地表明，同时测量热量产生和氧消耗速率可以确定氧化磷酸化的焓效率和ATP/O产率。然而，只有当与磷酸化系统相关的净焓变不为零时，这些测定才有可能。对于完整的酵母细胞，结果表明，同时进行热量产生和氧消耗速率的稳态测量可以评估焓生长效率（即与用于完全分解代谢和合成代谢的能量相比，作为生物质储存的能量）。该方法基于在纯有氧代谢过程中生长与静止条件下测定的量热-呼吸比（CR比）之间的比较。因此，与焓平衡方法不同，该方法不依赖于对代谢物和生物质元素组成的详尽且繁琐的测定。因此，可以在存在非限量碳底物的情况下进行实验，这种方法已成功应用于生长缓慢的细胞，如表达野生型或突变型大鼠解偶联蛋白-1的酵母细胞。
