One-step purification of Escherichia coli H(+)-ATPase (F0F1) and its reconstitution into liposomes with neurotransmitter transporters.
About 30% of the protein in the inner membrane of Escherichia coli strain DK8/pBWU13 is H(+)-ATPase (F0F1), and practically homogeneous F0F1 could be obtained by gradient centrifugation after solubilization of these membranes. The recombinant plasmid pBWU13 carries the unc operon for F0F1. When reconstituted into liposomes, F0F1 formed an ATP-dependent proton gradient and membrane potential. Proteoliposomes reconstituted with F0F1 and solubilized transporters from chromaffin granules or synaptic vesicle membranes could transport serotonin, dopamine, and norepinephrine dependent on ATP hydrolysis. F0F1 can be obtained rapidly from DK8/pBWU13, and its reconstitution into liposomes with transporters may be useful for monitoring these transporters during their purification.
Moriyama
Y
Y
Department of Organic Chemistry and Biochemistry, Osaka University, Japan.
Iwamoto
A
A
Hanada
H
H
Maeda
M
M
Futai
M
M
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D015306:Biogenic Monoamines / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient / Q000379:methods; D002837:Chromaffin Granules / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008081:Liposomes; D009876:Operon; D010957:Plasmids; D011510:Proteolipids; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*
D015306:Biogenic Monoamines; D002352:Carrier Proteins; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D006180:Proton-Translocating ATPases