Purification and properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa.
Purification and properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa.
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铜绿假单胞菌吡啶核苷酸转氢酶的纯化和性质。
DOI：
10.1016/s0021-9258(18)63064-9
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发表时间：
1970
期刊：
影响因子：
--
通讯作者：
Nathan O. Kaplan
中科院分区：
文献类型：
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作者：
Phillip T. Cohen;Nathan O. Kaplan
从铜绿假单胞菌（Pseudomonasaerodyniae）中分离纯化了一种2 ′-AMP激活的吡啶核苷酸转氢酶，并进行了结晶。该酶具有典型的黄素蛋白吸收光谱。通过厌氧添加TPNH，光谱部分漂白，并且漂白伴随着较长波长吸收的增加。随后加入DPN+逆转漂白。该酶催化TPNH和DPNH还原二氯吲哚酚和铁氰化钾，并且DPNH的这种还原被2 '-AMP激活。如果用纹影光学或测量黄素吸收的分光光度扫描装置监测分析超离心中的沉降，则观察到不存在2 '-AMP或TPN+时的非均相聚集体系和存在2'-AMP或TPN+时的明显均相体系。在TPNH存在下在51°下孵育酶或用酸性硫酸铵处理酶会导致活性损失。添加FAD而非黄素单甘肽可恢复活性。上述证据表明转氢酶是一种黄素蛋白，并提示天然辅因子是FAD。沉降速度实验表明，在观察的条件下，纯化的酶是非常大的。当浓度为8 mg/ml时，在2 '-AMP或TPN+存在下，转氢酶以ans 20，wof33.8沉淀。对羟基苯甲酸汞可引起酶的抑制和激活，底物可提供保护作用。2'-AMP激活还原底物不含2 '-磷酸基团的几个反应。当TPNH浓度约为3 × 10 ~（-4）ml时，还原底物不被2 ′-AMP激活。2 ′-AMP可提高DPNH对酶结合黄素的厌氧还原速率。这种效应在高浓度酶下观察到，可能反映了2 '-AMP分解酶的能力，但不一定反映了2'-AMP激活反应的机制，其中只有催化浓度的酶存在。所有测试的底物都是无活性的。
A 2'-AMP-activated pyridine nucleotide transhydrogenase fromPseudomonas aeruginosahas been purified to apparent homogeneity and crystallized. The enzyme has a typical flavoprotein absorption spectrum. The spectrum is partially bleached by the anaerobic addition of TPNH and the bleaching is accompanied by increases in longer wave length absorption. Subsequent addition of DPN+reverses the bleaching. The enzyme catalyzes reduction of dichloroindolphenol and potassium ferricyanide by TPNH and DPNH, and this reduction by DPNH is activated by 2'-AMP. If sedimentation in the analytical ultracentrifuge is monitored with schlieren optics or with a spectrophotometric scanning device measuring flavin absorption, a heterogeneous, aggregated system in the absence and an apparently homogenous system in the presence of 2'-AMP or TPN+are observed. Incubation of the enzyme at 51° in the presence of TPNH or treatment of the enzyme with acidic ammonium sulfate causes a loss of activity. Addition of FAD, but not flavin mononucleotide, restores activity. The above evidence indicates that the transhydrogenase is a flavoprotein and suggests that the natural cofactor is FAD.The sedimentation velocity experiments indicate that under the conditions of observation the purified enzyme is very large. At a concentration of 8 mg per ml and in the presence of 2'-AMP or TPN+the transhydrogenase sediments with ans20,wof 33.8.p-Hydroxymercuribenzoate can cause both inhibition and activation of the enzyme and protection against these effects is afforded by substrates.2'-AMP activates several reactions in which the reducing substrate does not contain a 2'-phosphate group. When TPNH at about 3x10-4mis the reducing substrate there is no activation by 2'-AMP.2'-AMP enhances the rate of anaerobic reduction of enzyme-bound flavin by DPNH. This effect was observed at high concentrations of enzyme and may reflect the ability of 2'-AMP to disaggregate the enzyme, yet not necessarily reflect the mechanism by which 2'-AMP activates reactions in which only catalytic concentrations of enzyme are present.The enzyme has been tested for its ability to catalyze reduction or oxidation of a large number of physiological, nonpyridine nucleotide substrates. All substrates tested were inactive.