Rowland Andrew, Gaganis Paraskevi, Elliot David J, Mackenzie Peter I, Knights Kathleen M, Miners John O
Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, SA 5042, Australia.
J Pharmacol Exp Ther. 2007 Apr;321(1):137-47. doi: 10.1124/jpet.106.118216. Epub 2007 Jan 19.
Studies were performed to elucidate the mechanism responsible for the reduction in Km values of UDP-glucuronosyltransferase 2B7 (UGT2B7) substrates observed for incubations conducted in the presence of albumin. Addition of bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSA-FAF), but not "crude" HSA, resulted in an approximate 90% reduction in the Km values for the glucuronidation of zidovudine (AZT) by human liver microsomes (HLM) and UGT2B7 and a 50 to 75% reduction in the S50 for 4-methylumbelliferone (4MU) glucuronidation by UGT2B7, without affecting Vmax. Oleic, linoleic, and arachidonic acids were shown to be the most abundant unsaturated long-chain fatty acids present in crude HSA and in the membranes of HLM and human embryonic kidney (HEK)293 cells, and it was demonstrated that these and other unsaturated long-chain fatty acids were UGT2B7 substrates. Glucuronides with Rf (retention factor) values corresponding to the glucuronides of linoleic and arachidonic acid were detected when HLM and HEK293 cell lysates were incubated with radiolabeled cofactor, and the intensity of the bands was modulated by the presence of crude HSA (increased) and BSA or HSA-FAF (decreased). Oleic, linoleic, and arachidonic acid inhibited AZT and 4MU glucuronidation by HLM and/or UGT2B7, due to an increase in Km/S50 without a change in Vmax. Addition of BSA and HSA-FAF reversed the inhibition. Likewise, coexpression of UGT2B7 and HSA in HEK293 cells reduced the Km/S50 values of these substrates. It is postulated that BSA and HSA-FAF sequester inhibitory fatty acids released during incubations, and the apparent high Km values observed for UGT2B7 substrates arise from the presence of these endogenous inhibitors.
开展了多项研究，以阐明在白蛋白存在下进行孵育时观察到的UDP-葡萄糖醛酸基转移酶2B7（UGT2B7）底物Km值降低的机制。添加牛血清白蛋白（BSA）和无脂肪酸人血清白蛋白（HSA-FAF），而非“粗制”HSA，可使人肝微粒体（HLM）和UGT2B7对齐多夫定（AZT）葡萄糖醛酸化的Km值降低约90%，UGT2B7对4-甲基伞形酮（4MU）葡萄糖醛酸化的S50降低50%至75%，且不影响Vmax。油酸、亚油酸和花生四烯酸是粗制HSA以及HLM和人胚肾（HEK）293细胞膜中最丰富的不饱和长链脂肪酸，并且已证明这些以及其他不饱和长链脂肪酸是UGT2B7的底物。当HLM和HEK293细胞裂解物与放射性标记的辅因子一起孵育时，检测到了Rf（保留因子）值与亚油酸和花生四烯酸葡萄糖醛酸化物相对应的葡萄糖醛酸化物，且条带强度受粗制HSA（增加）以及BSA或HSA-FAF（降低）的存在调节。油酸、亚油酸和花生四烯酸抑制HLM和/或UGT2B7对AZT和4MU的葡萄糖醛酸化，原因是Km/S50增加而Vmax不变。添加BSA和HSA-FAF可逆转这种抑制作用。同样，在HEK293细胞中共表达UGT2B7和HSA可降低这些底物的Km/S50值。据推测，BSA和HSA-FAF可隔离孵育过程中释放的抑制性脂肪酸，而观察到的UGT2B7底物明显较高的Km值源于这些内源性抑制剂的存在。
