Rees W D, Holman G D
Biochim Biophys Acta. 1981 Aug 20;646(2):251-60. doi: 10.1016/0005-2736(81)90331-x.
(1) The t 1/2 for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 +/- 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate (t 1/2 = 75.8 +/- 4.99 min) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are Koizt = 271.3 +/- 34.2 mM, Voizt = 1.15 +/- 0.12 mM . s-1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the Ki for a substrate analogue inhibitor should equal the equilibrium exchange Km for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the beta-position at C-1. The Ki for D-glucose = 8.62 mM; the Ki for beta-fluoro-D-glucose = 6.87 mM. Replacing the ring oxygen also results in a marked reduction in affinity. The Ki for 5-thio-D-glucose = 42.1 mM. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-D-galactose (Ki = 20.75 mM) has a slightly higher affinity than D-galactose (Ki = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as D-talose (Ki = 35.4 mM) has a lower affinity than D-galactose. (5) D-Allose (Km = 271.3 mM) and 3-deoxy-D-glucose (Ki = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The Ki for 3-fluoro-D-glucose = 7.97 mM. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-D-glucose (Ki = 11.08 mM) has lower affinity than D-glucose and 6-deoxy-D-galactose (Ki = 33.97 mM) has lower affinity than D-galactose. Fluorine solution at C-6 of D-galactose restores high affinity. The Ki for 6-fluoro-D-galactose = 6.67 mM. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The Ki for D-xylose = 45.5 mM. The Ki for L-arabinose = 49.69 mM. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.
(1) 在胰岛素刺激的脂肪细胞中，1.3 mM D-阿洛糖摄取和流出的t1/2为1.7±0.1分钟。在没有胰岛素的情况下，D-阿洛糖的介导摄取几乎被消除，摄取速率（t1/2 = 75.8±4.99分钟）接近非介导转运计算得出的速率。胰岛素处理细胞中D-阿洛糖零转运摄取的动力学参数为Koizt = 271.3±34.2 mM，Voizt = 1.15±0.12 mM·s-1。(2) 提出了与两种底物（或底物加抑制剂）相互作用的单门转运体（载体）模型的动力学分析。分析表明，与转运体相互作用的两种底物的异源交换速率并非唯一，可根据每种糖单独与转运体作用的动力学参数计算得出。这意味着底物类似物对低亲和力底物（如D-阿洛糖）转运抑制的方程可以简化。结果表明，对于单门转运体，底物类似物抑制剂的Ki应等于该类似物的平衡交换Km。(3) 在C-1处取代的类似物表明，融合的吡喃糖环被转运体接受。1-脱氧-D-葡萄糖可被转运，但对转运体的亲和力较低。通过在C-1的β位取代氟可恢复高亲和力。D-葡萄糖的Ki = 8.62 mM；β-氟-D-葡萄糖的Ki = 6.87 mM。取代环中的氧也会导致亲和力显著降低。5-硫代-D-葡萄糖的Ki = 42.1 mM。(4) C-2处具有葡糖构型的羟基不是必需的，因为2-脱氧-D-半乳糖（Ki = 20.75 mM）的亲和力略高于D-半乳糖（Ki = 24.49 mM）。C-2处具有甘露糖构型的羟基会干扰转运，因为D-塔罗糖（Ki = 35.4 mM）的亲和力低于D-半乳糖。(5) D-阿洛糖（Km = 271.3 mM）和3-脱氧-D-葡萄糖（Ki = 40.31 mM）的亲和力较低，但通过在C-3处取代葡糖构型的氟可恢复高亲和力。3-氟-D-葡萄糖的Ki = 7.97 mM。(6) 在C-4和C-6处修饰的类似物在亲和力上没有大幅损失。然而，6-脱氧-D-葡萄糖（Ki = 11.08 mM）的亲和力低于D-葡萄糖，6-脱氧-D-半乳糖（Ki = 33.97 mM）的亲和力低于D-半乳糖。D-半乳糖C-6处的氟取代可恢复高亲和力。6-氟-D-半乳糖的Ki = 6.67 mM。去除C-5羟甲基会导致亲和力大幅损失。D-木糖的Ki = 45.5 mM。L-阿拉伯糖的Ki = 49.69 mM。(7) 这些结果表明，参与糖与胰岛素刺激的脂肪细胞转运体相互作用的重要氢键位置是环中的氧、C-1和C-3。可能与C-6存在较弱的氢键。非葡糖构型的糖羟基可能在空间上阻碍转运。
