Fannin F F, Evans J O, Gibbs E M, Diedrich D F
Biochim Biophys Acta. 1981 Dec 7;649(2):189-201. doi: 10.1016/0005-2736(81)90406-5.
A new phloretin derivative, phloretinyl-3'-benzylazide (PBAz), has been synthesized and compared with phloretin for its ability to inhibit the hexose transporter in human erythrocyte membranes in subdued light. Transport measurements were made using the light scattering (Orskov optical) method and a Millipore filtration technique with isotopically labeled sugars. Initial rates of sugar flux were measured under four different conditions to test for inhibition asymmetry. In each experimental condition, PBAz is from 6-20-times more potent than phloretin, making it one of the most effective reversible inhibitors known. Although both agents penetrate the cell membrane, they apparently fail to reach inhibitory levels at the inner surface over the time course of our nonequilibrated experiments, because of extensive binding to hemoglobin. The mechanism by which PBAz and its parent phloretin inhibit transport is pure competition with hexose for the carrier which faces the exterior of the membrane. If given time to equilibrate with the cells, the inhibition by both agents converts to a mixed type, i.e., both competitive and noncompetitive. The noncompetitive component could be due to inhibition of those transporter units oriented internally. Alternatively pre-equilibration with the inhibitors may cause them to attain high levels in the lipid membrane and produce nonspecific effects. PBAz and its precursor amine, phloretinyl-3'-benzylamine (PBA), compete with glucose for the sugar binding site on mutarotase at least as well as phloretin. When exposed to long wavelength ultraviolet radiation, PBAz is converted to a reactive intermediate which becomes covalently bound to the enzyme. Both irreversible ligand attachment and mutarotase inhibition are related to dose of the azide and irradiation time, but inactivation is from 5 to 6-times greater than label incorporation. We conclude that PBAz is a potentially useful photoaffinity labeling agent capable of covalently interacting with the transporter site facing the exterior of the red cell.
一种新的根皮素衍生物，根皮素-3'-苄基叠氮化物（PBAz）已被合成，并与根皮素在弱光条件下抑制人红细胞膜中己糖转运蛋白的能力进行了比较。使用光散射（奥斯科夫光学）方法和带有同位素标记糖的密理博过滤技术进行转运测量。在四种不同条件下测量糖通量的初始速率，以测试抑制的不对称性。在每种实验条件下，PBAz的效力比根皮素高6至20倍，使其成为已知最有效的可逆抑制剂之一。尽管两种试剂都能穿透细胞膜，但在我们的非平衡实验过程中，由于它们与血红蛋白大量结合，显然未能在内表面达到抑制水平。PBAz及其母体根皮素抑制转运的机制是与己糖对面向膜外的载体进行纯竞争。如果给予时间使其与细胞平衡，两种试剂的抑制作用都会转变为混合型，即既有竞争性又有非竞争性。非竞争性成分可能是由于对内部取向的那些转运蛋白单元的抑制。或者，与抑制剂预平衡可能导致它们在脂质膜中达到高水平并产生非特异性效应。PBAz及其前体胺，根皮素-3'-苄胺（PBA），与葡萄糖竞争变旋酶上的糖结合位点，其竞争能力至少与根皮素一样好。当暴露于长波长紫外线辐射时，PBAz会转化为一种反应性中间体，该中间体与酶形成共价结合。不可逆配体附着和变旋酶抑制都与叠氮化物的剂量和照射时间有关，但失活比标记掺入大5至6倍。我们得出结论，PBAz是一种潜在有用的光亲和标记剂，能够与面向红细胞外部的转运蛋白位点发生共价相互作用。
