Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A(2) activities.
Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A(2) activities.
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DOI：
10.1016/j.abb.2009.02.008
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发表时间：
2009-05-15
影响因子：
3.9
通讯作者：
Fisher AB
中科院分区：
文献类型：
--
作者：
Manevich Y;Shuvaeva T;Dodia C;Kazi A;Feinstein SI;Fisher AB
Peroxiredoxin 6（Prdx 6）不同于其他哺乳动物peroxiredoxins在其能力，以减少磷脂氢过氧化物在中性pH值和在具有磷脂酶A2（PLA 2）的活动，是最大的酸性pH值。我们以前显示了一个活性位点C47过氧化物酶活性和催化三联体S32-H26-D140必需的磷脂和PLA 2活性的结合。本研究评估了在胞浆pH下还原和氧化磷脂氢过氧化物与Prdx 6的结合。将重组Prdx 6与1-棕榈酰-2-亚油酰-sn-甘油-3-磷酸胆碱氢过氧化物（PLPCOOH）孵育导致过氧化物酶活性，如用Prdx 6-SO2（3）抗体检测到的cys 47氧化，和通过圆二色性分析的Prdx 6熔融温度的显著偏移，表明PLPCOOH是Prdx 6的特异性底物。通过DNS-PE或bis-Pyr荧光的变化和超滤检测Prdx 6与氧化脂质体的优先结合。Prdx 6中S32或H26的位点特异性突变消除了结合，而D140突变没有影响。用过氧化物处理A549细胞导致脂质过氧化和Prdx 6从胞质溶胶易位到细胞膜。因此，Prdx 6的两种酶活性的pH特异性可以通过蛋白质的差异结合动力学来解释; Prdx 6在酸性pH下与还原的磷脂结合，但在胞质pH下仅与氧化的磷脂结合，这与Prdx 6在修复过氧化细胞膜中的作用相容。
Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A2 (PLA2) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA2 activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO2(3) antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.
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DOI：
10.1016/s0305-0491(98)10046-9
发表时间：
1998-06-01
影响因子：
2.2
作者：
Akiba, S;Dodia, C;Fisher, AB
通讯作者：
Fisher, AB
DOI：
10.1152/ajplung.1998.274.5.l750
发表时间：
1998-05-01
影响因子：
4.9
作者：
Kim, TS;Dodia, C;Fisher, AB
通讯作者：
Fisher, AB
影响因子：
4.8
作者：
Kang, SW;Baines, IC;Rhee, SG
通讯作者：
Rhee, SG
DOI：
10.1073/pnas.182384499
发表时间：
2002-09-03
影响因子：
11.1
作者：
Manevich, Y;Sweitzer, T;Fisher, AB
通讯作者：
Fisher, AB
影响因子：
4.8
作者：
Pak, JH;Manevich, Y;Fisher, AB
通讯作者：
Fisher, AB