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首页> 外文期刊>Biochemistry >Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.
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Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.

机译：UDP-葡萄糖醛酸转移酶同工酶在大鼠肝微粒体内的蛋白相互作用。

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摘要

The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for direct interaction of UGT2B1 with UGT1s was obtained by the loss of UGT2B1 adsorption to immunoaffinity column in Gunn rat hepatic microsomes, which lack all UGT1 isozymes. When the microsomes were treated with the chemical cross-linking reagent 1,6-bis(maleimido)-hexane, a cross-linked product with an apparent molecular mass of 120-130 kDa was obtained that immunostained with antibodies against UGT1s and UGT2B1, indicating the formation of a heterodimer containing one of the UGT1 isozymes and UGT2B1. The effects of UGT complex formation on the stimulation of glucuronidation of testosterone and uptake of UDP-glucuronic acid (UDP-GlcUA) by UDP-N-acetylglucosamine (UDP-GlcNAc) were examined. Alkaline pH-induced dissociation of the complexes was associated with the loss of UDP-GlcNAc-dependent stimulation of glucuronidation, suggesting that two functional states of UGTs with different kinetic parameters correspond to the monomer and oligomer form of UGTs in the membranes. The UDP-GlcNAc-dependent stimulation of UDP-GlcUA uptake into the microsomal vesicles also was affected by the extent of complex formation. These results suggest that complex formation of the UGT isozymes affects the UDP-GlcNAc-dependent stimulation of glucuronidation via stimulation of UDP-GlcUA uptake.
机译：使用具有抗肽抗体的免疫纯化技术和化学交联策略，研究了大鼠肝微粒体中UDP-葡萄糖醛酸糖基转移酶（UGT）同工酶UGT1s和UGT2B1之间的相互作用。通过氨基末端测序和抗UGT2B1的抗肽抗体进行免疫检测，将经UGT1s共免疫纯化的50 kDa蛋白鉴定为UGT2B1。在缺少所有UGT1同工酶的耿恩大鼠肝微粒体中，UGT2B1吸附到免疫亲和柱上的损失获得了UGT2B1与UGT1s直接相互作用的证据。当用化学交联剂1,6-双（马来酰亚胺基）-己烷处理微粒体时，获得了表观分子量为120-130 kDa的交联产物，并用针对UGT1s和UGT2B1的抗体进行了免疫染色，表明形成含有UGT1同工酶和UGT2B1之一的异二聚体。研究了UGT复合物形成对刺激睾丸激素葡萄糖醛酸化和UDP-N-乙酰氨基葡萄糖（UDP-GlcNAc）摄取UDP-葡萄糖醛酸（UDP-GlcUA）的影响。碱性pH诱导的复合物的解离与UDP-GlcNAc依赖性葡萄糖醛酸苷化刺激的丧失有关，这表明具有不同动力学参数的UGT的两个功能状态对应于膜中UGT的单体和低聚物形式。 UDP-GlcNAc依赖UDP-GlcUA摄取到微粒体小泡中的刺激也受到复合物形成程度的影响。这些结果表明，UGT同工酶的复杂形成会通过刺激UDP-GlcUA摄取来影响UDP-GlcNAc依赖性葡萄糖醛酸苷化刺激。

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