Interaction between pyridine adenine dinucleotides and bovine liver catalase: a chromatographic and spectral study.
Interaction between pyridine adenine dinucleotides and bovine liver catalase: a chromatographic and spectral study.
复制标题
吡啶腺嘌呤二核苷酸和牛肝过氧化氢酶之间的相互作用：色谱和光谱研究。
DOI：
10.1016/0003-9861(86)90402-9
复制
发表时间：
1986
影响因子：
3.9
通讯作者：
J. Gaillard
中科院分区：
文献类型：
--
作者：
H. Jouve;J. Pelmont;J. Gaillard
结晶牛肝过氧化氢酶中存在两个不同的组分，并且可以使用染料-配体亲和色谱与含有Procion HE 3B的Red-A Matrex凝胶进行分离。大部分（α）未吸附在该凝胶上。第二组分（β）牢固地吸附在凝胶上，并且可以被高盐或微摩尔范围内的NADPH洗脱。过氧化氢酶β在较高浓度的NADH、NADP+和ADP也能得到洗脱。组分α和β在比活性、热稳定性和光吸收数据方面未显示出可检测的差异。这表明α和β之间的行为差异与NADPH与哺乳动物过氧化氢酶的结合有关[H. N. Kirkman和G. F. Gaetani（1984）Proc. Natl. Acad. Sci. USA 81，4343-4347]，并且β部分对应于具有至少一个用于NADPH结合的游离位点的酶分子。利用光吸收和EPR数据研究了二硫代赤藓醇（DTE）存在下过氧化氢酶分子的修饰。硫醇诱导的变化，对应于形成的过氧化氢酶复合物II。在很低的NADPH水平下，它们被部分逆转，并且二核苷酸似乎在此过程中被氧化。DTE处理过氧化氢酶在Red-A Matrex柱上完全吸附，并以β组分洗脱。在DTE和NADPH存在下，奇异变形杆菌的过氧化氢酶也显示出类似的光谱变化。该酶也能够氧化NADPH，但不被Red-A Matrex吸附。这项工作表明，染料亲和层析提供了一个非常方便的工具，用于分离二核苷酸耗尽的过氧化氢酶从牛肝，促进进一步研究的酶内的这种辅因子的生理功能。
Two different fractions were present in crystalline bovine liver catalase, and could be resolved using dye-ligand affinity chromatography with Red-A Matrex gel containing Procion HE3B. The major part (α) was not adsorbed on this gel. The second fraction (β) was firmly adsorbed to the gel, and could be eluted either by high salt or by NADPH in the micromolar range. Elution of catalase β was also obtained with NADH, NADP+, and ADP at higher concentration. Fractions α and β displayed no detectable difference in specific activity, stability to heat, and light absorption data. It is suggested that the difference in behavior between α and β is related to the binding of NADPH to the mammalian catalase [H. N. Kirkman and G. F. Gaetani (1984)Proc. Natl. Acad. Sci. USA81, 4343–4347], and that the β fraction corresponds to the enzyme molecules that have at least one free site for NADPH binding. Modifications of catalase molecules in the presence of dithioterythritol (DTE) were examined using light absorption and EPR data. Thiol induced changes that corresponded to the formation of catalase complex II. They were partially reversed by NADPH at very low level, and the dinucleotide appeared to be oxidized in this process. DTE-treated bovine catalase was totally adsorbed on the Red-A Matrex columns, and could be eluted as fraction β. Similar spectral changes in the presence of DTE and NADPH were displayed by a bacterial catalase fromProteus mirabilis. This enzyme was also able to oxidize NADPH, but was not adsorbed by Red-A Matrex. This work suggests that dye-affinity chromatography provides a very convenient tool for isolating dinucleotide-depleted catalase from bovine liver, facilitating further study of the physiological function of this cofactor within the enzyme.