Yamaguchi K, Hosokawa Y, Kohashi N, Kori Y, Sakakibara S, Ueda I
J Biochem. 1978 Feb;83(2):479-91. doi: 10.1093/oxfordjournals.jbchem.a131935.
Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.
大鼠肝脏半胱氨酸双加氧酶已被纯化至同质。它是一种单亚基蛋白，分子量为22,500±1,000，等电点为5.5。纯化得到的酶无催化活性，通过与L-半胱氨酸或其类似物如羧甲基-L-半胱氨酸、羧乙基-L-半胱氨酸、S-甲基-L-半胱氨酸、D-半胱氨酸、半胱胺、N-乙酰-L-半胱氨酸和DL-高半胱氨酸进行厌氧孵育而被激活。用L-半胱氨酸激活的酶在有氧条件下迅速失活。在0℃时观察到这种快速失活，此时未检测到反应产物半胱亚磺酸或半胱氨酸的自氧化产物胱氨酸的形成。进一步分析表明，激活酶的失活是由于氧气，但与底物的存在、酶的周转或测定过程中产生的抑制剂的积累无关。一种独特的大鼠肝脏细胞质蛋白，称为蛋白A，可以完全防止酶的有氧失活。在没有蛋白A的情况下测定过程中活性的丧失被证明是一个一级衰变过程。从log（Δ产物/分钟）对时间的图中，获得了初始速度（V0）和7分钟时的速度（V7）。根据初始速度计算出在没有蛋白A的情况下L-半胱氨酸的表观Km值为4.5×10^(-4)M。蛋白A没有改变L-半胱氨酸的表观Km值。当在预激活前加入邻菲罗啉、α，α'-联吡啶、bathophenanthroline、8-羟基喹啉、EGTA和EDTA等螯合剂时，它们会强烈抑制酶的活性。纯化的半胱氨酸双加氧酶每摩尔酶蛋白含有1个铁原子。通过激活程序，该酶对热变性、螯合剂的抑制作用和胰蛋白酶消化的敏感性降低。
