Brizio C, Otto A, Brandsch R, Passarella S, Barile M
Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Italy.
Eur J Biochem. 2000 Jul;267(14):4346-54. doi: 10.1046/j.1432-1327.2000.01464.x.
The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.
对大鼠肝脏线粒体参与线粒体基质黄素酶二甲基甘氨酸脱氢酶（Me2GlyDH）的黄素化作用进行了研究。Me2GlyDH在兔网织红细胞裂解物（RL）转录/翻译系统中作为脱辅基酶合成，其黄素化通过全酶对胰蛋白酶的抗性来监测。在添加FAD的情况下，通过加入溶解的线粒体、线粒体基质和DEAE纯化的基质组分，全酶形成速率以递增的效率受到刺激。当溶解的线粒体补充FMN和ATP时，脱辅基Me2GlyDH也转化为全酶。这一观察结果与存在一种线粒体FAD合成酶相一致，该酶从前体生成全酶形成所需的FAD。在存在FAD的情况下，全酶形成与测定中基质蛋白的浓度呈线性增加，并取决于外部添加的Me2GlyDH的量，具有饱和特性。这些发现表明线粒体基质中存在一种刺激Me2GlyDH黄素化的蛋白质因子。免疫去除实验表明，该因子不同于线粒体热休克蛋白（Hsp）70，而不含Hsp60的DEAE纯化基质组分加速RL翻译和纯化的Me2GlyDH的黄素化能力表明，该因子也不同于线粒体Hsp60。
