Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase.
His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.
Ishmukhametov
Robert R
RR
Department of Biological Sciences, PO Box 750376, 6501 Airline Road, Southern Methodist University, Dallas, Texas 75275-0376, USA.
Galkin
Mikhail A
MA
Vik
Steven B
SB
D000609:Aminoacridines / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D010957:Plasmids / Q000235:genetics; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*
D000609:Aminoacridines; D011510:Proteolipids; C020485:proteoliposomes; C010847:9-amino-6-chloro-2-methoxyacridine; D006639:Histidine; D006180:Proton-Translocating ATPases