Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes.
Incorporation of a bacterial membrane-bound hydrogenase into proteoliposomes.
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将细菌膜结合氢化酶掺入蛋白脂质体中。
DOI：
10.1016/0003-2697(92)90308-t
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发表时间：
1992
影响因子：
2.9
通讯作者：
Maier,RJ
中科院分区：
文献类型：
--
作者：
Ferber,DM;Maier,RJ
来自不同属细菌的膜结合镍铁氢化酶以前已经被表征，它们是密切相关的。我们报道了纯化的缓生根瘤菌日本氢化酶重组成蛋白脂质体的洗涤剂透析方法，然后进行两到三个周期的冻融。沉淀实验表明，当重组成大肠杆菌磷脂时，超过60%的h2摄取活性是颗粒状的。蔗糖梯度离心将氢化酶活性分离成两个峰，其中密度较低的峰与磷脂相关且浑浊，因此表明成功结合。纯化后的酶不能与预先形成的磷脂囊泡结合，1.0 m NaCl不能去除加入的氢化酶。氢化酶掺入胶束洗涤剂的最佳磷脂比（ϱ）值为2.0。含有酸性磷脂的蛋白脂质体在结合和活性方面都是最有效的。人工电子受体的特异性与日本血吸虫蛋白脂质体和h2氧化膜相似。在最佳条件下形成的蛋白脂质体具有以直径400 nm为中心的较宽的尺寸分布。蛋白质修饰试剂重氮苯磺酸盐（DABS）可部分保护蛋白脂质体中的氢化酶活性（失活t1 2= 30 min），而DABS可快速失活纯化酶（t1 2= 4 min）。后一个结果表明磷脂双分子层保护了一个重要的催化位点。该实验系统应有助于解决有关细菌膜结合氢化酶在体内情况的问题。
Membrane-bound nickel-iron hydrogenases from diverse genera of bacteria have been previously characterized and they are closely related. We report the reconstitution of purified Bradyrhizobium japonicum hydrogenase into proteoliposomes by a detergent dialysis method followed by two or three cycles of freezethaw. Sedimentation experiments revealed that more than 60% of the H2-uptake activity was particulate when reconstituted into Escherichia coli phospholipids. Sucrose-gradient centrifugation separated hydrogenase activity into two peaks, the less dense of which was phospholipid-associated and turbid, thereby showing successful incorporation. Purified enzyme did not bind to preformed phospholipid vesicles, and 1.0 m NaCl failed to remove incorporated hydrogenase. The optimal micellar detergent:phospholipid ratio (ϱ) value for hydrogenase incorporation was 2.0. Proteoliposomes containing acidic phospholipids were the most effective for incorporation as well as for activity. The artificial electron acceptor specificity was similar for proteoliposomes and for H2-oxidizing membranes from B. japonicum. Proteoliposomes formed under optimal conditions had a broad size distribution centered around 400 nm diameter. Hydrogenase activity in proteoliposomes was partially protected from inactivation by the protein modification reagent diazobenzene sulfonate (DABS) (inactivation t1 2= 30 min , whereas DABS rapidly inactivated the purified enzyme (t1 2= 4 min . The latter result indicates protection of a catalytically important site by the phospholipid bilayer. This experimental system should be useful in addressing questions regarding the in vivo situation of bacterial membrane-bound hydrogenases.
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DOI：
--
发表时间：
1982
期刊：
影响因子：
--
作者：
R. McElhaney
通讯作者：
R. McElhaney
DOI：
--
发表时间：
1971
期刊：
Canadian Journal of Microbiology (print)
影响因子：
--
作者：
C. Bunn;G. H. Elkan
通讯作者：
G. H. Elkan
亲和层析有氧纯化日本根瘤菌氢化酶
影响因子：
3.2
作者：
L. Stults;F. Moshiri;R. Maier
通讯作者：
R. Maier
DOI：
--
发表时间：
1983
期刊：
影响因子：
--
作者：
S. Mutaftschiev;M. R. O'Brian;R. Maier
通讯作者：
R. Maier
将免疫球蛋白 E 受体重建到脂质体中。
影响因子：
4.8
作者：
B. Rivnay;H. Metzger
通讯作者：
H. Metzger