Pineda T, Churchich J E
Department of Biochemistry, University of Tennessee, Knoxville 37916.
J Biol Chem. 1993 Sep 25;268(27):20218-22.
The unfolding of brain pyridoxal kinase by guanidinium HCl has been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after removal of guanidinium HCl. Unfolding of pyridoxal kinase was monitored by circular dichroism and fluorescence spectroscopy. The steepness of the spectroscopic changes between 0.2 and 1.5 M guanidinium HCl, and the lack of any discernible plateau suggests that unfolding of the monomer is a cooperative process. A compact intermediate on the unfolding pathway of pyridoxal kinase could not be detected by the method of denaturant gel filtration. The fluorescent analogs of the substrates ATP and pyridoxal were used to assess differences in stability among the domains of the protein. Based on fluorescence and steady emission anisotropy results, it is postulated that the nucleotide domain is more stable than the pyridoxal domain of the kinase.
已在平衡状态下研究了盐酸胍对脑吡哆醛激酶的去折叠作用。从去除盐酸胍后催化活性的完全恢复判断，整个过程是可逆的。通过圆二色性和荧光光谱监测吡哆醛激酶的去折叠。在0.2至1.5 M盐酸胍之间光谱变化的陡峭程度，以及缺乏任何可辨别的平稳期，表明单体的去折叠是一个协同过程。通过变性剂凝胶过滤法未检测到吡哆醛激酶去折叠途径上的紧密中间体。底物ATP和吡哆醛的荧光类似物用于评估蛋白质各结构域之间稳定性的差异。基于荧光和稳态发射各向异性结果，推测激酶的核苷酸结构域比吡哆醛结构域更稳定。
