Hutchinson J P, el-Thaher T S, Miller A D
Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, U.K.
Biochem J. 1994 Sep 1;302 ( Pt 2)(Pt 2):405-10. doi: 10.1042/bj3020405.
In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.
在大肠杆菌伴侣蛋白cpn60（groEL）和cpn10（groES）存在的情况下，研究了猪线粒体苹果酸脱氢酶的体外重折叠。当该酶最初用3M氯化胍变性时，伴侣蛋白辅助的重折叠效率为100%。圆二色光谱显示，苹果酸脱氢酶在3M氯化胍中几乎完全展开，这表明几乎没有或没有残留二级结构的状态是伴侣蛋白辅助重折叠的最佳“底物”。事实证明，变性为更高结构状态的苹果酸脱氢酶在伴侣蛋白的辅助下重折叠效率较低。结果表明，该酶在重折叠条件下不会聚集，因此重折叠效率的损失是由不可逆的错误折叠造成的。有证据表明，伴侣蛋白无法挽救不可逆错误折叠的苹果酸脱氢酶。利用100K Centricon浓缩器通过超滤结合试验研究了[14C]乙酰基标记的苹果酸脱氢酶与groEL的结合。通过Scatchard图分析数据表明，先前已用氯化胍广泛展开的乙酰基苹果酸脱氢酶在特定结合位点与groEL结合。在饱和状态下，一个乙酰基苹果酸脱氢酶同型二聚体（两条多肽链）与每个groEL同型寡聚体结合，结合常数约为10 nM。
