在缺乏镁离子或钙离子刺激的三磷酸腺苷酶的大肠杆菌K12突变株的膜中恢复能量偶联转氢酶活性。

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase.

作者信息

Cox G B, Gibson F, McCann L M, Butlin J D, Crane F L

出版信息

Biochem J. 1973 Apr;132(4):689-95. doi: 10.1042/bj1320689.

DOI:10.1042/bj1320689

PMID:4269101

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1177644/

Abstract

We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 2. The Mg(2+),Ca(2+)-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA(-)) could not be re-activated by the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA(-)).

摘要

我们分离出了大肠杆菌K12（菌株AN295）的一个突变体，该突变体形成了去阻遏量的Mg(2+)、Ca(2+)刺激的腺苷三磷酸酶。2. 通过用含有EDTA和二硫苏糖醇的5mM Tris-HCl缓冲液提取，将菌株AN295膜制剂中的Mg(2+)、Ca(2+)刺激的三磷酸酶活性分离出来，导致ATP依赖性转氢酶活性丧失。非能量偶联的转氢酶活性保留在膜残渣中。3. 通过反复凝胶过滤对菌株AN295中溶解的Mg(2+)、Ca(2+)刺激的腺苷三磷酸酶活性进行部分纯化。将纯化的Mg(2+)、Ca(2+)刺激的腺苷三磷酸酶添加到菌株AN295的膜残渣中，可重新激活ATP依赖性转氢酶活性。4. 菌株AN296缺乏Mg(2+)、Ca(2+)刺激的腺苷三磷酸酶活性，是通过将突变等位基因uncA401转导到菌株AN295中获得的。ATP依赖性转氢酶活性丧失，但非能量偶联的转氢酶得以保留。5. 菌株AN296（uncA(-)）膜制剂中的ATP依赖性转氢酶活性不能被菌株AN295纯化的Mg(2+)、Ca(2+)刺激的腺苷三磷酸酶重新激活。然而，在用含有EDTA和二硫苏糖醇的5mM Tris-HCl缓冲液提取后，通过向菌株AN296（uncA(-)）的膜残渣中添加菌株AN295纯化的Mg(2+)、Ca(2+)刺激的腺苷三磷酸酶，ATP依赖性转氢酶活性可以被重新激活。