Subunit c from the sodium-ion-translocating F1F0-ATPase of Propionigenium modestum--production, purification and properties of the protein in dodecylsulfate solution.
Escherichia coli strain PEF42 produces a sodium-ion-dependent hybrid F1F0-ATPase consisting of the Propionigenium modestum subunits a, b, c and delta, of a hybrid alpha subunit and of the E. coli subunits beta, gamma and epsilon. The gene encoding subunit c of the P. modestum F1F0-ATPase was cloned into the pT7-7 expression vector to yield plasmid pT7c. E. coli PEF42 was transformed with plasmid pT7c together with plasmid pGP1-2, which harbours the gene for the T7 RNA polymerase. The production of the P. modestum subunit c was induced by a temperature shift from 30 degrees C to 42 degrees C for 30 min and led to an increased concentration of this protein in the membrane of the host strain. The c subunit produced in E. coli moved as a monomer in dodecylsulfate electrophoresis. The protein was extracted from the cells with chloroform/methanol, purified and incorporated into sodium dodecylsulfate micelles. Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature-stable spectrum (between 20-60 degrees C) with a high proportion of alpah-helical structure. Upon incubation of subunit c with [14C]dicyclohexylcarbodiimide the protein became labelled in a sodium-ion-dependent manner, similar to the labelling observed if the purified F1F0-ATPase of P. modestum, was treated with the radioactive carbodiimide. The Na+-specific site was therefore retained in the isolated c subunit dissolved in dodecylsulfate.
Matthey
U
U
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Kaim
G
G
Dimroth
P
P
D001421:Bacteria, Anaerobic / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D017136:Ion Transport; D008823:Micelles; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012964:Sodium / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012996:Solutions; D013329:Structure-Activity Relationship
D008823:Micelles; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; D004024:Dicyclohexylcarbodiimide; D012964:Sodium; D006180:Proton-Translocating ATPases