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Adipose gene expression response of lean and obese mice to short-term dietary restriction.瘦小鼠和肥胖小鼠对短期饮食限制的脂肪基因表达反应。
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MALIC ENZYME AND LIPOGENESIS.苹果酸酶与脂肪生成
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Crystallographic studies on Ascaris suum NAD-malic enzyme bound to reduced cofactor and identification of an effector site.猪蛔虫NAD-苹果酸酶与还原型辅因子结合的晶体学研究及效应位点的鉴定。
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Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate.ATP和富马酸酯对人线粒体NAD(P)+依赖性苹果酸酶的调控分子机制。
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Crystal structure of the malic enzyme from Ascaris suum complexed with nicotinamide adenine dinucleotide at 2.3 A resolution.猪蛔虫苹果酸酶与烟酰胺腺嘌呤二核苷酸复合物的晶体结构,分辨率为2.3埃。
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人胞质苹果酸酶二聚体和四聚体界面相互作用的生物物理特性分析。

Biophysical characterization of the dimer and tetramer interface interactions of the human cytosolic malic enzyme.

机构信息

Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan.

出版信息

PLoS One. 2012;7(12):e50143. doi: 10.1371/journal.pone.0050143. Epub 2012 Dec 21.

DOI:10.1371/journal.pone.0050143
PMID:23284632
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3528742/
Abstract

The cytosolic NADP(+)-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8-10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.

摘要

细胞质 NADP(+)-依赖性苹果酸酶 (c-NADP-ME) 具有二聚体-二聚体四级结构,其中二聚体界面的结合比四聚体界面更紧密。在这项研究中,使用荧光和圆二色性光谱、分析超速离心和酶活性监测 c-NADP-ME 界面突变体的尿素诱导展开过程。在这里,我们证明了人 c-NADP-ME 二聚体和四聚体界面相互作用之间的差异蛋白稳定性。我们的数据清楚地表明,c-NADP-ME 的蛋白质稳定性主要受到二聚体界面而不是四聚体界面的破坏的影响。首先,在热稳定性实验中,野生型和四聚体界面突变体的熔点比二聚体界面突变体高 8-10°C。其次,在尿素变性实验中,野生型和四聚体界面突变体的热力学参数几乎相同。然而,对于二聚体界面突变体,尿素展开曲线的第一个转变向较低的尿素浓度移动,并且展开中间物在较低的尿素浓度下存在。第三,对于四聚体 WT c-NADP-ME,在 2 M 尿素处理之前,酶首先从四聚体解离为二聚体,然后二聚体在 2.5 M 尿素处理之前解离为单体。对于二聚体四聚体界面突变体 (H142A/D568A),在 2.5 M 尿素处理后,二聚体完全解离为单体,而对于二聚体二聚体界面突变体 (H51A/D90A),在 1.5 M 尿素处理后,二聚体完全解离为单体,表明 c-NADP-ME 在二聚体界面的相互作用确实比在四聚体界面更强。因此,这项研究为为什么苹果酸酶需要组装为二聚体的二聚体提供了合理的解释。