Peng Yang, Kumar Sanath, Hernandez Ricardo L, Jones Suzanna E, Cadle Kathleen M, Smith Kenneth P, Varela Manuel F
Department of Biology, Eastern New Mexico University, Portales, NM 88130, USA.
J Membr Biol. 2009 Mar;228(2):79-88. doi: 10.1007/s00232-009-9161-9. Epub 2009 Mar 18.
The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure "downhill" maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation ("uphill" transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport, and permease structure should consider maltose, as well as sucrose.
本研究的目的是检测来自大肠杆菌的次级主动转运蛋白蔗糖通透酶（CscB）的糖识别和转运特性。我们验证了野生型CscB转运蛋白具有麦芽糖转运功能这一假设。携带pSP72/cscB的大肠杆菌HS4006细胞在麦芽糖麦康凯琼脂指示平板上呈红色。我们能够检测到“顺浓度梯度”的麦芽糖转运，并确定此类细胞中麦芽糖进入的明确动力学行为。在具有CscB的细胞中，麦芽糖是蔗糖转运的有效竞争者，这表明通过CscB通道的各自麦芽糖和蔗糖结合位点及转运途径相互重叠。具有CscB的细胞对麦芽糖的积累（“逆浓度梯度”转运）很显著，证明CscB能主动转运麦芽糖。用于转运研究的细胞中所使用的质粒pSP72/cscB上编码的cscB测序表明CscB的一级结构未改变，排除了突变赋予CscB麦芽糖转运功能的可能性。我们得出结论，麦芽糖是大肠杆菌蔗糖通透酶的真正底物。因此，未来关于糖结合、转运和通透酶结构的研究应同时考虑麦芽糖和蔗糖。
