tailieunhanh - Báo cáo Y học: Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits

TheatpBandatpF genes ofPropionigenium modestumwere cloned as His-tag fusion constructs and expressed in Escherichia recombinant subunits a and b were purified via Ni 2+ chelate affinitychromatography. A func-tionallyactive Focomplex was reassembledin vitro from subunits a, band c, and incorporated into liposomes. | Eur. J. Biochem. 269 2567-2573 2002 FEBS 2002 doi Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits Franziska Wehrle Yvonne Appoldt Georg Kaim and Peter Dimroth Institut fur Mikrobiologie Eidgenõssische Technische Hochschule Zurich Switzerland The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni2 chelate affinity chromatography. A lucc-tionally ccti ve Fo complex was reassembled in vitro from subunits a b and c and incorporated into liposomes. The Fo liposomes catalysed 22Na uptake in response to an inside negative potassium diffusion potential and the uptake was prevented by mochhictiómi of die c Sbbnniss with N N -dicyclohexylcarbodiimide DCCD . In the absence of a membrane potential the Fo complexes catalysed 22Na out Na in-exchange. After F1 addition the F1Fo complex was formed and the holoenzyme catalysed ATP synthesis ATP dependent Na pumping and ATP hydrolysis which was inhibited byDCCD. Functional F o hybrids were reconstituted with recombinant subunits a and b from P. eoodestim and c11 from Ilyobacter tartaricus. These Fo hybrids had Na translocation activities that were not distinguishable from that of P. modesuum Fo. Keywords ATP synthase Fo reconstitution Na translocation subunit a subunit b. F1Fo type ATP synthases are widely distributed among eukaryotes plants and bacteria. Utilizing the energy stored in an electrochemical ion gradient these enzymes catalyse the synthesis of ATP from ADP and inorganic phosphate. In bacteria the enzyme can also operate in reverse as an ATP-driven ion pump 1-3 . Detailed structural knowledge is available for the water-soluble F1 headpiece with the subunit composition a3b3yỗe. Alternating a and b subunits form a cylinder around subunit y. Part of the y subunit .

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