Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at

硫酸铵与盐酸胍对鸡肝二氢叶酸还原酶的激活和失活作用

在５℃、２０℃、３０℃，鸡肝二氢叶酸还原酶的活力随盐酸孤浓度增加，先经历一个激活区，以后则为失活区。温度升高，使最大激活幅度变小，激活区变窄，所需盐酸孤浓度减小。硫酸铵在浓度低于０．２ｍｏｌ／Ｌ时，对天然酶有较小的激活作用，更高浓度的硫酸按使酶活力下降；胍激活酶的活力随硫酸铵浓度的增加，由天然酶的２１０％单调下降；对失活酶，增加硫酸铵的浓度，则使酶活力逐渐恢复；三种情形的终活力均为天然酶的４０％。相应条件下的荧光光谱和圆二色谱的结果表明，硫酸铵对天然酶与激活酶的构象影响不大，但可使失活酶的构象恢复到与天然酶相似的状态。