West S M, Price N C
School of Molecular and Biological Sciences, University of Stirling, Scotland, U.K.
Biochem J. 1989 Jul 1;261(1):189-96. doi: 10.1042/bj2610189.
The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred.
研究了猪心细胞质天冬氨酸转氨酶在氯化胍(GdnHCl)溶液中的展开情况。蛋白质荧光、圆二色性和巯基反应活性的数据表明,该酶在6M - GdnHCl中发生了展开。光谱研究表明,这种展开伴随着磷酸吡哆醛辅因子的解离。在稀释GdnHCl时,只要存在二硫苏糖醇和磷酸吡哆醛,酶就会以合理的产率重新激活。活性的恢复遵循二级动力学。在没有添加二硫苏糖醇和磷酸吡哆醛的情况下,会大量形成高分子量聚集体。