Schröter A, Kopperschläger G
Institut für Biochemie, Universitätsklinikum, Universität Leipzig, Germany.
FEMS Microbiol Lett. 1996 Sep 1;142(2-3):247-52. doi: 10.1111/j.1574-6968.1996.tb08438.x.
6-Phosphofructo-1-kinase (PFK, EC 2.7.1.11) activity was detected in extracts of Rhodotorula glutinis grown on different nitrogen and carbon sources. The activity of PFK varied depending on the composition of the culture medium and on the state of growth. Using a carbon-limited medium containing glucose and a mixed organic/inorganic nitrogen source, maximal yield, activity and stability of PFK were found under aerobic conditions at the end of the exponential growth phase. Native Rhodotorula-PFK could be separated by polyacrylamide gel electrophoresis. Applying this method for molecular mass estimation a value of 450 +/- 90 kDa was calculated. Taking into account a molecular mass of 130 +/- 5 kDa for the subunit, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following immunoblotting, a tetrameric structure of the native PFK is likely. Polyclonal antibodies, raised against PFK from Saccharomyces cerevisiae, were able to cross-react with Rhodotorula-PFK. Therefore, there are similarities in the primary structure of both enzymes. Fructose 2,6-bisphosphate was identified as a significant activator of Rhodotorula-PFK leading to a 10-fold activation of the enzyme: maximal activation was achieved with 5 microM fructose 2,6-bisphosphate.
在生长于不同氮源和碳源上的粘红酵母提取物中检测到了6-磷酸果糖-1-激酶（PFK，EC 2.7.1.11）的活性。PFK的活性因培养基组成和生长状态而异。使用含有葡萄糖和有机/无机混合氮源的碳限制培养基，在指数生长期末期的有氧条件下发现了PFK的最大产量、活性和稳定性。天然的粘红酵母PFK可以通过聚丙烯酰胺凝胶电泳分离。应用这种方法进行分子量估计，计算出的值为450±90 kDa。考虑到通过免疫印迹后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定的亚基分子量为130±5 kDa，天然PFK可能具有四聚体结构。针对酿酒酵母PFK产生的多克隆抗体能够与粘红酵母PFK发生交叉反应。因此，这两种酶的一级结构存在相似性。果糖2,6-二磷酸被鉴定为粘红酵母PFK的一种重要激活剂，可使该酶激活10倍：在5 microM果糖2,6-二磷酸时实现最大激活。
