Verstappen R, Ranostaj S, Rausch T
Botanisches Institut, Johann Wolfgang Goethe Universität, Frankfurt F.R.G.
Biochim Biophys Acta. 1991 Mar 4;1073(2):366-73. doi: 10.1016/0304-4165(91)90144-6.
The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent Km value of 230 microM (substrate: 3-O-methyl-D-glucose (3-OMG); pHi 7.2/pHo 7.2), which was reduced to 120 microM when a pH gradient was imposed (pHo 5.7/pHi 7.2). However, the Vmax value was not affected indicating that no stable pH gradient was formed. Uptake experiments with 14C-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500 microM), whereas the tonoplast hexose transporter (in mixed inside / out and outside / out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent Km values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.
利用膜泡进行跨零转运研究，对异养生长的转基因烟草细胞的质膜己糖转运体和液泡膜己糖转运体进行了体外研究。在高度纯化的、经相分离的外翻质膜囊泡（PMV）中，己糖转运体对3-O-甲基-D-葡萄糖（3-OMG）的表观Km值为230μM（底物；pHi 7.2/pHo 7.2），当施加pH梯度（pHo 5.7/pHi 7.2）时，该值降至120μM。然而，Vmax值未受影响，这表明未形成稳定的pH梯度。用14C标记的乙酸盐进行的摄取实验支持了这一解释。转运对N-乙基马来酰亚胺（NEM；浓度高达1 mM）和对氯汞苯磺酸盐（PCMBS；浓度高达500μM）不敏感，而液泡膜己糖转运体（在混合的内翻/外翻和外翻/外翻囊泡中）以底物保护的方式被NEM抑制，PCMBS也具有抑制作用。对于液泡膜己糖转运体，在动力学上可区分出表观Km值分别为6和20 mM的两个组分。除D-半乳糖和D-果糖外，两种转运体的底物特异性相似。结果表明植物液泡膜和质膜己糖转运体之间存在结构差异。
