Miyagi Yukino, Matsumura Yoshihiro, Sagami Hiroshi
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1, Katahira, Sendai, Japan.
J Biochem. 2007 Sep;142(3):377-81. doi: 10.1093/jb/mvm144. Epub 2007 Jul 23.
A recombinant geranylgeranyl diphosphate synthase (GGPS) was analysed to be a mixture of octamer, hexamer and dimer by gel filtration using a Superdex 200 column followed by the blue native polyacrylamide gel electrophoresis. The hexamer and dimer were each converted to an octamer by treating with dithiothreitol (DTT). When the recombinant GGPS was preliminarily treated with DTT and similarly analysed, octamer was predominantly detected with a trace amount of hexamer. The octameric form of GGPS was also supported by the cross-linking experiments with bis(sulfosuccinimidyl) suberate. The GGPS in an octameric form was active with a combination of farnesyl diphosphate and [1-(14)C]isopentenyl diphosphate. These results indicate that the active form of GGPS in the solution is an octamer rather than hexamer or dimer.
通过使用Superdex 200柱进行凝胶过滤，随后进行蓝色天然聚丙烯酰胺凝胶电泳分析，发现重组香叶基香叶基二磷酸合酶（GGPS）是八聚体、六聚体和二聚体的混合物。通过用二硫苏糖醇（DTT）处理，六聚体和二聚体各自转化为八聚体。当重组GGPS用DTT进行预处理并进行类似分析时，主要检测到八聚体，伴有微量的六聚体。用双（磺基琥珀酰亚胺基）辛二酸酯进行的交联实验也支持GGPS的八聚体形式。八聚体形式的GGPS与法呢基二磷酸和[1-（14）C]异戊烯基二磷酸结合时具有活性。这些结果表明，溶液中GGPS的活性形式是八聚体，而不是六聚体或二聚体。
