Jiang R F, Wang Z X, Xu G J
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Biochim Biophys Acta. 1997 Nov 14;1343(1):95-101. doi: 10.1016/s0167-4838(97)00125-8.
The unfolding and refolding behavior of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach in solutions of guanidine hydrochloride (GdnHCl) was studied. By a number of criteria (enzyme activity, protein fluorescence, circular dichroism), the enzyme was judged to be almost completely unfolded in 6 M GdnHCl. The changes in enzyme activity occur at lower concentrations of GdnHCl than those required to bring about changes in circular dichroism (CD) and fluorescence, as has been found for other enzymes. Spinach Rubisco is completely inactive in 0.5 M GdnHCl with no apparent changes observed in the overall structure of the enzyme as monitored by CD and intrinsic fluorescence. The result of the size-exclusion chromatography indicates that the inactive enzyme still exists in the hexadecameric state. On dilution of the GdnHCl, reactivation of the inactive enzyme by low concentrations of GdnHCl occurred. The regain of activity was time-dependent and obeyed first-order kinetics, and the substrate, ribulose-1,5-biphosphate, can stimulate this reactivation process. The result suggests that the inactivation of the Rubisco in dilute GdnHCl is caused by the conformational changes at the active site instead of the inhibition of guanidine hydrochloride or the dissociation of the oligomeric enzyme molecules.
研究了菠菜中十六聚体核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）在盐酸胍（GdnHCl）溶液中的去折叠和重折叠行为。通过多种标准（酶活性、蛋白质荧光、圆二色性）判断，该酶在6 M GdnHCl中几乎完全去折叠。与其他酶一样，酶活性的变化发生在比引起圆二色性（CD）和荧光变化所需浓度更低的GdnHCl浓度下。菠菜Rubisco在0.5 M GdnHCl中完全无活性，通过CD和内在荧光监测，未观察到酶的整体结构有明显变化。尺寸排阻色谱结果表明，无活性的酶仍以十六聚体状态存在。在稀释GdnHCl时，低浓度的GdnHCl会使无活性的酶重新激活。活性的恢复与时间相关且符合一级动力学，底物核酮糖-1,5-二磷酸可刺激这一重新激活过程。结果表明，稀GdnHCl中Rubisco的失活是由活性位点的构象变化引起的，而不是盐酸胍的抑制作用或寡聚酶分子的解离。
