Chen J, Smith D L
Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Protein Sci. 2001 May;10(5):1079-83. doi: 10.1110/ps.53201.
Although there is general agreement that native mitochondrial malate dehydrogenase (MDH) exists as a dimer at pH 7, its aggregation state at pH 5 is less certain. The present amide hydrogen exchange study was performed to determine whether MDH remains a dimer at pH 5. To detect pH-induced changes in solvent accessibility, MDH was exposed to D(2)O at pH 5 or 7, then fragmented with pepsin into peptides that were analyzed by mass spectrometry. Even after adjustments for the effect of pH on the intrinsic rate of hydrogen exchange, large increases in deuterium levels were found at pH 5 only in peptic fragments derived from the subunit binding surface of MDH. In parallel experiments, elevated deuterium levels were also found in the same regions of MDH monomer trapped inside a mutant form of the chaperonin GROEL: This selective increase in hydrogen exchange rates, which was attributed to increased solvent accessibility of these regions, provides new evidence that MDH is a monomer at pH 5.
虽然人们普遍认为天然线粒体苹果酸脱氢酶（MDH）在pH 7时以二聚体形式存在，但其在pH 5时的聚集状态尚不确定。进行本酰胺氢交换研究以确定MDH在pH 5时是否仍为二聚体。为了检测pH诱导的溶剂可及性变化，将MDH置于pH 5或7的重水中，然后用胃蛋白酶裂解成肽段，通过质谱分析。即使对pH对氢交换内在速率的影响进行了校正，仅在源自MDH亚基结合表面的胃蛋白酶消化片段中发现pH 5时氘水平大幅升高。在平行实验中，被困在伴侣蛋白GROEL突变形式内的MDH单体的相同区域也发现氘水平升高：这种氢交换速率的选择性增加归因于这些区域溶剂可及性的增加，为MDH在pH 5时是单体提供了新证据。
