Vanstapel F, Blanckaert N
J Biol Chem. 1987 Apr 5;262(10):4616-23.
Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.
天然胆红素（BR）的共轭作用依赖于一种肝脏微粒体UDP-糖基转移酶，该酶使用UDP-葡萄糖（UDP-Glc）、UDP-木糖，且主要使用UDP-葡萄糖醛酸（UDP-GlcA）。我们发现，当将源自大鼠或豚鼠肝脏的洗涤过的完整微粒体与BR在不添加UDP-糖的情况下孵育时，BR会发生酯化反应。这种内源性酯化反应主要导致BR单葡萄糖苷的两种位置异构体的形成，并且显示出与用饱和浓度的添加UDP-Glc孵育的微粒体形成的BR单葡萄糖苷相同的区域选择性。这一发现以及缺乏BR UDP-糖基转移酶活性的突变大鼠肝脏微粒体中不存在内源性酯化反应，证明内源性酯化反应依赖于UDP-糖基转移酶，因此表明UDP-Glc存在于完整的微粒体囊泡中。当向微粒体外孵育培养基中添加UDP-Glc时，在用去污剂、超声处理或金黄色葡萄球菌α-毒素预处理微粒体破坏膜通透性屏障后，BR的葡萄糖苷化显著增强。相反，这种膜破坏导致BR的内源性酯化反应消失，并且发现内源性酯化反应的受损与囊泡破坏程度之间存在直接关系，这表明内源性酯化反应所依赖的UDP-Glc存在于微粒体的腔隙空间中。动力学证据以及增加微粒体中多萜醇-P-葡萄糖（Dol-P-Glc）浓度对内源性酯化反应无影响，排除了Dol-P-Glc直接或间接参与内源性酯化反应的可能性。在37℃而非0℃下，将完整微粒体与UDP-Glc或UDP-木糖预孵育，会导致内源性酯化反应所依赖的微粒体UDP-糖池扩大，这表明两种UDP-糖都可以通过温度依赖性机制进入微粒体囊泡。与这些发现相反，当微粒体在37℃下与UDP-GlcA预孵育时，未检测到BR酯化反应增加。我们得出结论，天然完整的微粒体含有腔内源性UDP-Glc池，并且BR UDP-葡萄糖基转移酶和UDP-木糖基转移酶由于其腔内取向，可直接接触假定的微粒体内核苷酸糖池。
