O'Gorman E, Fuchs K H, Tittmann P, Gross H, Wallimann T
Institute for Cell Biology, ETH Honggerberg, Zurich, Switzerland.
J Cell Sci. 1997 Jun;110 ( Pt 12):1403-11. doi: 10.1242/jcs.110.12.1403.
Rats were fed a 2% guanidino propionic acid diet for up to 18 weeks to induce cellular creatine depletion by inhibition of creatine uptake by this creatine analogue. Ultrastructural analysis of creatine depleted tissues showed that mitochondrial intermembrane inclusion bodies appeared in all skeletal muscles analysed, after 11 weeks of feeding. Heart had relatively few even after 18 weeks of analogue feeding and none were evident in kidney, brain or liver. These structures were strongly immuno-positive for sarcomeric mitochondrial creatine kinase and upon removal from mitochondria, the inclusion bodies were shown to diffract to a resolution of 2.5 nm. Two-dimensional image analysis and three-dimensional reconstruction revealed arrays of creatine kinase octamers with additional components between the octameric structures. The same mitochondria had a 3-fold higher extractable specific creatine kinase activity than controls. Molecular mass gel filtration of inclusion body containing mitochondrial extracts from analogue fed rat solei revealed mitochondrial creatine kinase eluting as an aggregate of an apparent molecular mass > or = 2,000 kDa. Mitochondrial creatine kinase of control soleus mitochondrial extract eluted as an octamer, with a molecular mass of 340 kDa. Respiration measurements of control solei mitochondria displayed creatine mediated stimulation of oxidative phosphorylation that was absent in analogue-fed rat solei mitochondria. The latter also had 19% and 14% slower rates of state 4 and maximal state 3 respiration, respectively, than control mitochondria. These results indicate that mitochondrial creatine kinase co-crystallises with another component within the inter membrane space of select mitochondria in creatine depleted skeletal muscle, and is inactive in situ.
给大鼠喂食含2%胍基丙酸的饮食长达18周，通过这种肌酸类似物抑制肌酸摄取来诱导细胞内肌酸耗竭。对肌酸耗竭组织的超微结构分析表明，喂食11周后，在所有分析的骨骼肌中均出现线粒体内膜包涵体。即使在喂食类似物18周后，心脏中的包涵体相对较少，而在肾脏、大脑或肝脏中均未发现明显的包涵体。这些结构对肌节线粒体肌酸激酶呈强免疫阳性，从线粒体中取出后，包涵体显示出2.5纳米的衍射分辨率。二维图像分析和三维重建揭示了肌酸激酶八聚体阵列，在八聚体结构之间还有其他成分。相同的线粒体可提取的比肌酸激酶活性比对照高3倍。对喂食类似物的大鼠比目鱼肌线粒体提取物进行的分子量凝胶过滤显示，线粒体肌酸激酶以表观分子量>或 = 2000 kDa的聚集体形式洗脱。对照比目鱼肌线粒体提取物的线粒体肌酸激酶以分子量为340 kDa的八聚体形式洗脱。对照比目鱼肌线粒体的呼吸测量显示，肌酸介导的氧化磷酸化刺激在喂食类似物的大鼠比目鱼肌线粒体中不存在。后者的状态4呼吸速率和最大状态3呼吸速率分别比对照线粒体慢19%和14%。这些结果表明，线粒体肌酸激酶在肌酸耗竭的骨骼肌中与特定线粒体内膜间隙中的另一种成分共结晶，并且在原位无活性。
