Zhi W, Landry S J, Gierasch L M, Srere P A
Department of Veterans Affairs Medical Center, Dallas, Texas 75216.
Protein Sci. 1992 Apr;1(4):522-9. doi: 10.1002/pro.5560010407.
Citrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants--bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol--promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586-1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, < 5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE-assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA-assisted refolding, this system will be useful in future studies on the mechanism of GroE-facilitated refolding.
柠檬酸合酶（CS），其在盐酸胍（GdnHCl）或尿素中变性后，可以通过多种方式辅助复性。添加每种辅助剂——牛血清白蛋白（BSA）、草酰乙酸（OAA）和甘油——都能促进复性。这些物质共同作用时，对折叠态CS的产率具有加和效应。Buchner等人（Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586 - 1591）报道GroE系统（一种由GroEL [cpn60]和GroES [cpn10]组成的大肠杆菌伴侣蛋白[cpn]）能促进CS的重折叠，这一报道已得到证实。然而，我们观察到重新激活的CS产率显著更高，为82%，且在没有GroES时几乎没有重新激活，<5%，而Buchner等人分别报道为28%和16%。此外，我们发现GroE辅助的重折叠对在GdnHCl中变性的CS比在尿素中变性的CS更有效。根据已知的在GdnHCl和尿素中产生的变性状态差异对这一结果进行了讨论。由于GroEL抑制BSA/甘油/OAA辅助的重折叠，该系统将有助于未来对GroE促进重折叠机制的研究。
