Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli
Sequence differences between human muscle and liver cDNAs for UDPglucose pyrophosphorylase and kinetic properties of the recombinant enzymes expressed in Escherichia coli
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DOI：
10.1111/j.1432-1033.1996.00173.x
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发表时间：
1996-01-15
期刊：
影响因子：
--
通讯作者：
Chang, HY
中科院分区：
文献类型：
--
作者：
Duggleby, RG;Chao, YC;Chang, HY
UDP-Glc 焦磷酸化酶 (EC 2.7.7.9) 催化 MgUTP 加 Glc1P 和 UDP-Glc 加 MgPPi 的相互转化。对缺乏这种活性的大肠杆菌菌株进行补充，可以从人类肌肉文库中分离出编码这种酶的 cDNA。鉴定出两种形式并确定了每种形式的核苷酸序列；我们发现它们仅在 5' 区域存在差异，我们认为这些差异是由于在两个转录本中使用了不同的第一个外显子所致。这些核苷酸序列与先前从人肝脏文库中分离的 cDNA 的序列不同[Peng，H.-L. & 张，H.-Y。 (1993) 费用快报。 329, 153-158]并且提出这些肝脏和肌肉农场源自不同的基因。肌肉形式 I、肌肉形式 II、肝脏形式和与部分 lacZ 基因融合的肝脏形式的 cDNA 在大肠杆菌中表达，并表征了每种酶的动力学特性。肌肉形式 I 和 LacZ/肝脏融合酶对所有底物都表现出 Michaelis-Menten 动力学，而肌肉形式 II 的速率对 MgPPi 浓度呈 S 型依赖性。肝脏形式显示出 MgUTP 的 Michaelis-Menten 动力学。对于其余三种底物，观察到复杂的动力学，涉及低底物浓度下的乙状结肠性和高底物浓度下的部分抑制的组合。
UDP-Glc pyrophosphorylase (EC 2.7.7.9) catalyses the interconversion of MgUTP plus Glc1P and UDP-Glc plus MgPPi. Complementation of an Escherichia coli strain lacking this activity has allowed isolation of cDNA encoding this enzyme from a human muscle library. Two forms were identified and the nucleotide sequence of each was determined; they were found to differ only in the 5' region and we suggest that these arise from the use of a different first exon in the two transcripts. These nucleotide sequences are different from that of the cDNA which was isolated previously from a human liver library [Peng, H.-L. & Chang, H.-Y. (1993) FEES Lett. 329, 153-158] and it is proposed that these liver and muscle farms are derived from different genes. The cDNA for muscle form I, muscle form II, the liver form, and the liver form fused to part of the lacZ gene were expressed in Escherichia coli and the kinetic properties of each enzyme were characterised, Muscle form I and the LacZ/liver fusion enzyme exhibit Michaelis-Menten kinetics towards all substrates while muscle form II has a sigmoidal dependence of rate upon the concentration of MgPPi. The liver form shows Michaelis-Menten kinetics towards MgUTP. For the remaining three substrates, complex kinetics were observed involving a combination of sigmoidicity at low substrate concentration and partial inhibition at high substrate concentration.