Roossien F F, Robillard G T
Biochemistry. 1984 Nov 20;23(24):5682-5. doi: 10.1021/bi00319a003.
The association state of the mannitol-specific enzyme II (EIIMtl) has been studied both in the purified form and embedded in the cytoplasmic membrane. Membrane fragments obtained from mannitol-grown Escherichia coli catalyze the phosphoenolpyruvate- (PEP) dependent phosphorylation of both glucose and mannitol; thus they contain both the glucose- and mannitol-specific enzymes II. The autoradiogram of an electrophoresed mixture of [32P]PEP, EI, HPr, and membrane fragments shows bands at 58 and 116 kilodaltons, in addition to the bands of P-EI and P-HPr. In an analogous experiment with purified EIIMtl, suspended in detergent micelles, only a 58 000-dalton band and the P-HPr and P-EI bands were found. Treatment of the phosphorylated membranes with mannitol results in an immediate substantial decrease in the radioactivity in the 58- and 116-kilodalton bands. A similar treatment of the phosphorylated membranes with glucose had no direct effect on the autoradiogram. We conclude therefore that the 58- and 116-kilodalton bands originate from enzyme IIMtl monomers and dimers, respectively. The interaction between the subunits of the dimer is not abolished by the addition of up to 5% sodium dodecyl sulfate. However, the nonionic detergent Lubrol PX, which is present during the purification of EIIMtl, is capable of transforming the enzyme IIMtl dimers into monomers.
已对甘露醇特异性酶 II（EIIMtl）的缔合状态进行了研究，研究对象既有纯化形式的，也有嵌入细胞质膜中的。从以甘露醇为碳源生长的大肠杆菌中获得的膜片段可催化磷酸烯醇丙酮酸（PEP）依赖的葡萄糖和甘露醇磷酸化反应；因此，它们同时含有葡萄糖特异性和甘露醇特异性的酶 II。[32P]PEP、EI、HPr 和膜片段的电泳混合物的放射自显影片，除了 P-EI 和 P-HPr 的条带外，还显示出 58 和 116 千道尔顿处的条带。在用悬浮于去污剂胶束中的纯化 EIIMtl 进行的类似实验中，仅发现了一条 58000 道尔顿的条带以及 P-HPr 和 P-EI 的条带。用甘露醇处理磷酸化的膜，会导致 58 和 116 千道尔顿条带中的放射性立即大幅下降。用葡萄糖对磷酸化的膜进行类似处理，对放射自显影片没有直接影响。因此，我们得出结论，58 和 116 千道尔顿的条带分别源自酶 IIMtl 的单体和二聚体。二聚体亚基之间的相互作用不会因添加高达 5%的十二烷基硫酸钠而被消除。然而，在纯化 EIIMtl 过程中存在的非离子去污剂 Lubrol PX 能够将酶 IIMtl 二聚体转化为单体。
