Kleinzeller A, McAvoy E M, McKibbin R D
Biochim Biophys Acta. 1980 Aug 4;600(2):513-29. doi: 10.1016/0005-2736(80)90453-8.
The active transport of methyl beta-D-galactoside and some other analogs of D-glucose and D-galactose was studied in slices of rabbit and renal cortex. 1. The non-metabolizable methyl beta-D-galactoside accumulates in renal cortical cells against its concentration gradient. At 1 mM substrate concentration (O2, 35 degrees C, 60 min incubation) the gradient was 2.36 +/- 0.11 S.E. (n = 33). The Kt was 1.50 +/- 0.02 mM. The active transport of the substrate was inhibited by dinitrophenol, phlorizin, absence of Na+ and by ouabain. This inhibition was incomplete, suggesting that the sugar may enter the cells by two separate pathways, only one of which was coupled to the down-hill electrochemical Na gradient. 2. The structural requirements for the interaction between substrate and the carrier were defined: (a) by testing the transport behavior of some analogs (1,5-anhydro-D-glucitol; methyl beta-thio-D-galadtoside; 3-deoxy-D-glucose; 4-deoxy-D-glucose; 5-thio-D-glucose; 6-deoxy-D-glucose and methyl-alpha-6-deoxy-D-glucoside); and (b) by inhibition analysis of methyl beta-D-galactoside transport. The role of each hydroxyl of the sugar molecule was tested by using a total of 41 analogs modified on each C by replacing -OH by -H, -O-CH3, -F and in some instances also by -SH. 3. The carrier is shared by D-glucose, D-galactose and their methyl glycosides. A pyranose ring is mandatory. The D-glucoconfiguration is preferred for the interaction with the carrier. 4. Replacement of -OH by -H or -S practically abolished (on C1, C2, C3) or greatly reduced (on C4) the affinity of the analog for the carrier. This was also confirmed by demonstrating that 1-deoxy-, and 3-deoxy-glucose and the thio-galactoside were not actively transported and their entry into the cells was not markedly affected by phlorizin, dinitrophenol, ouabain or absence of Na+. 4-Deoxy-D-glucose was taken up and its transport was inhibited by agents affecting the transport of methyl beta-D-galactoside. 5. Replacement of -OH by -F did not abolish the affinity of the analogs for the carrier, indicating hydrogen bonding between the carrier and the oxygens at C1, C2, C3, and C4. 6. 5-Thio-D-glucose was not transported against its concentration gradient and also poorly interacted with the carrier as shown by inhibition analysis. Hydrogen bonding between the carrier and the pyranose ring oxygen is suggested. 7. 6-Deoxyglucose is a potent inhibitor of methyl beta-D-galactoside transport although it is not actively taken up by the tissue. It is concluded that a hydroxyl at C6 is required for transport, but is not mandatory for an interaction with the carrier. However, 6-deoxy-D-galactose was ineffective as inhibitor. 8. The specificity of the carrier involved in the renal active transport of D-glucose, D-galactose and their methyl glycosides resembles qualitatively, and mostly also quantitatively that described for intestinal transport of these sugars.
在兔肾皮质切片中研究了β-D-甲基半乳糖苷以及D-葡萄糖和D-半乳糖的其他一些类似物的主动转运。1. 不可代谢的β-D-甲基半乳糖苷在肾皮质细胞中逆浓度梯度积累。在底物浓度为1 mM（O₂，35℃，孵育60分钟）时，浓度梯度为2.36±0.11标准误（n = 33）。米氏常数（Kt）为1.50±0.02 mM。底物的主动转运受到二硝基苯酚、根皮苷、无Na⁺以及哇巴因的抑制。这种抑制不完全，表明糖可能通过两条独立途径进入细胞，其中只有一条途径与下坡电化学Na⁺梯度偶联。2. 确定了底物与载体相互作用的结构要求：(a) 通过测试一些类似物（1,5-脱水-D-葡萄糖醇；β-D-硫代甲基半乳糖苷；3-脱氧-D-葡萄糖；4-脱氧-D-葡萄糖；5-硫代-D-葡萄糖；6-脱氧-D-葡萄糖和α-6-脱氧-D-葡萄糖甲基苷）的转运行为；(b) 通过对β-D-甲基半乳糖苷转运的抑制分析。通过总共41种类似物测试了糖分子每个羟基的作用，这些类似物在每个碳原子上通过将-OH替换为-H、-O-CH₃、-F，在某些情况下还替换为-SH进行了修饰。3. 载体由D-葡萄糖、D-半乳糖及其甲基糖苷共用。吡喃糖环是必需的。D-葡萄糖构型更有利于与载体相互作用。4. 将-OH替换为-H或-S实际上消除了（在C1、C2、C3上）或大大降低了（在C4上）类似物与载体的亲和力。通过证明1-脱氧-和3-脱氧-葡萄糖以及硫代半乳糖苷不被主动转运且它们进入细胞不受根皮苷、二硝基苯酚、哇巴因或无Na⁺的明显影响，这一点也得到了证实。4-脱氧-D-葡萄糖被摄取且其转运受到影响β-D-甲基半乳糖苷转运的试剂的抑制。5. 将-OH替换为-F并没有消除类似物与载体的亲和力，表明载体与C1、C2、C3和C4上的氧之间存在氢键。6. 5-硫代-D-葡萄糖不逆浓度梯度转运，并且如抑制分析所示与载体的相互作用也很差。提示载体与吡喃糖环氧之间存在氢键。7. 6-脱氧葡萄糖是β-D-甲基半乳糖苷转运的有效抑制剂，尽管它不被组织主动摄取。得出结论，C6上的羟基是转运所必需的，但不是与载体相互作用所必需的。然而，6-脱氧-D-半乳糖作为抑制剂无效。8. 参与D-葡萄糖、D-半乳糖及其甲基糖苷肾主动转运的载体的特异性在质量上相似，并且在数量上大多也与这些糖在肠道转运中所描述的相似。
