Haddaoui E A, Leloup L, Petit-Glatron M F, Chambert R
Institut Jacques Monod, Laboratoire Génétique et Membranes, Centre National de la Recherche Scientifique, Université Paris 7, France.
Eur J Biochem. 1997 Oct 15;249(2):505-9. doi: 10.1111/j.1432-1033.1997.00505.x.
Bacillus subtilis exocellular alpha-amylase is reversibly refolded after denaturation by guanidine hydrochloride at pH 7 and 37 degrees C. The unfolding-folding transition monitored by intrinsic fluorescence changes and resistance to proteolysis was resolved into a two-state transition. The first step (t1/2 < 1 s) led from D, the totally unfolded state, to C, a stable partially structured state of the protein. This folding intermediate was devoid of any enzyme activity and partially resistant to protease degradation. Calcium was required for the transition from C to N, the native state. This metal did not remain associated with the native form and could be replaced by barium or strontium, but not by magnesium. We discuss the hypothesis that C, the folding intermediate whose further transformation is under kinetic control, is the competent state involved in the secretion process of alpha-amylase.
枯草芽孢杆菌胞外α-淀粉酶在pH 7和37℃下被盐酸胍变性后可进行可逆重折叠。通过内在荧光变化和对蛋白酶解的抗性监测的去折叠-折叠转变被解析为双态转变。第一步（t1/2 < 1 s）从完全去折叠状态D转变为蛋白质的稳定部分结构化状态C。这种折叠中间体没有任何酶活性，并且对蛋白酶降解具有部分抗性。从C态转变为天然态N需要钙。这种金属不会与天然形式保持结合，并且可以被钡或锶取代，但不能被镁取代。我们讨论了这样一种假说，即C作为其进一步转化受动力学控制的折叠中间体，是参与α-淀粉酶分泌过程的活性状态。
