Bush D R
Photosynthesis Research Unit, U.S. Department of Agriculture, Illinois.
Arch Biochem Biophys. 1993 Dec;307(2):355-60. doi: 10.1006/abbi.1993.1600.
Sucrose transporters are important components of the assimilate partitioning pathway in many plants. In the results reported here, we examined the effect of several inhibitors on proton-coupled sucrose transport into plasma membrane vesicles isolated from sugar beet leaf tissue. Three compounds that are reversible inhibitors of glucose transporters, phlorizin, cytochalasin B, and forskolin, also inhibited the proton-sucrose symport. Additionally, several reagents that covalently modify specific amino acid residues, including p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide (NEM), diethyl pyrocarbonate (DEPC), and Hg2+, were also examined. NEM was not an effective inhibitor of the symport under both energized (pH 6.0) and unenergized (pH 7.7) conditions. In contrast, PCMBS, DEPC, and Hg2+ blocked sucrose transport activity. However, in control experiments it was discovered that Hg2+, but not PCMBS or DEPC, dissipated the proton electrochemical potential difference (delta mu H) that drives sucrose accumulation. It was further demonstrated that Hg2+ dissipated an imposed delta mu+H in protein-free liposomes, thus obscuring its effect on the sucrose symport. In time- and concentration-dependent inactivation experiments, it was shown that DEPC binding was substrate protectable, thereby implicating binding at or near the active site of the carrier. In contrast, PCMBS activity was not linked to substrate binding. DEPC activity was partially reversed with hydroxylamine. This is consistent with specific modification of a histidine residue. Preloading purified vesicles with free histidine did not slow the DEPC-dependent inactivation kinetics. Since these membrane vesicles are predominantly right-side out, the last observation is consistent with a DEPC-sensitive site which is accessible from the outside face of the vesicle. The results with DEPC suggest that a histidine residue is at or near the active site of the sucrose symport and that this amino acid plays a critical role in the reaction mechanism.
蔗糖转运蛋白是许多植物同化物分配途径的重要组成部分。在本文报道的结果中，我们研究了几种抑制剂对质子偶联的蔗糖转运进入从甜菜叶组织分离的质膜囊泡的影响。三种作为葡萄糖转运蛋白可逆抑制剂的化合物，根皮苷、细胞松弛素B和福斯可林，也抑制质子 - 蔗糖同向转运。此外，还研究了几种共价修饰特定氨基酸残基的试剂，包括对氯汞苯磺酸（PCMBS）、N - 乙基马来酰亚胺（NEM）、焦碳酸二乙酯（DEPC）和Hg²⁺。在有能量供应（pH 6.0）和无能量供应（pH 7.7）的条件下，NEM都不是同向转运的有效抑制剂。相比之下，PCMBS、DEPC和Hg²⁺阻断了蔗糖转运活性。然而，在对照实验中发现，Hg²⁺而非PCMBS或DEPC，耗散了驱动蔗糖积累的质子电化学势差（ΔμH）。进一步证明，Hg²⁺使无蛋白脂质体中施加的Δμ⁺H耗散，从而掩盖了其对蔗糖同向转运的影响。在时间和浓度依赖性失活实验中，结果表明DEPC结合可被底物保护，从而表明其在载体活性位点或附近结合。相比之下，PCMBS的活性与底物结合无关。DEPC的活性可被羟胺部分逆转。这与组氨酸残基的特异性修饰一致。用游离组氨酸预加载纯化的囊泡并没有减缓DEPC依赖性失活动力学。由于这些膜囊泡主要是外翻的，最后的观察结果与一个可从囊泡外表面接近的DEPC敏感位点一致。DEPC的结果表明，一个组氨酸残基位于蔗糖同向转运的活性位点或附近，并且该氨基酸在反应机制中起关键作用。
