Consalvi V, Millevoi S, Chiaraluce R, de Rosa M, Scandurra R
Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università La Sapienza, Roma, Italy.
Biochem Mol Biol Int. 1995 Feb;35(2):397-407.
The hexameric NAD-dependent glutamate dehydrogenase from the thermophilic eubacterium Bacillus acidocaldarius is the first glutamate dehydrogenase which spontaneously refolds in vitro. After 24 h unfolding in 6 M guanidinium chloride at 20 degrees C, refolding was achieved by dilution of the denaturant. The yield of reconstitution obtained in the presence of 1,4 dithio-DL-threitol in the unfolding/refolding mixture was 75%. The unfolding/refolding equilibria have been studied by fluorescence, circular dichroism and catalytic activity, which was 50% inhibited at 0.08 M guanidinium chloride, a value 30-fold lower than the transition midpoint detected by physical changes. Refolding was attempted in the presence of various additives, at different temperatures and varying enzyme and residual guanidinium chloride concentration. The refolded enzyme, after removal of inactive aggregated species, completely resembles the native enzyme in term of its physicochemical and kinetic properties as well as thermophilicity.
嗜热真细菌嗜酸热芽孢杆菌的六聚体NAD依赖型谷氨酸脱氢酶是首个能在体外自发重折叠的谷氨酸脱氢酶。在20℃下于6M盐酸胍中展开24小时后，通过稀释变性剂实现重折叠。在展开/重折叠混合物中存在1,4-二硫代-DL-苏糖醇时，重构产率为75%。通过荧光、圆二色性和催化活性研究了展开/重折叠平衡，在0.08M盐酸胍时催化活性被抑制50%，该值比通过物理变化检测到的转变中点低30倍。尝试在不同添加剂存在下、不同温度以及不同酶和残留盐酸胍浓度条件下进行重折叠。去除无活性聚集物后的重折叠酶在其物理化学和动力学性质以及嗜热性方面与天然酶完全相似。
