Chromatographic purification of the chloroplast ATP synthase (CF0-CF1) and the role of CF0 subunit IV in proton conduction.
Chromatographic procedures were developed to purify chloroplast ATP synthase (CF0-CF1) in large amounts and to resolve subunits from this enzyme. The ATP synthase thus obtained has high ATP-Pi exchange and Mg2(+)-ATPase activities upon incorporation into asolectin liposomes. The purity of this preparation was about 95%. By modifications of this chromatographic procedure, we purified subunit IV-deficient CF0-CF1, subunit IV-deficient CF0, and subunit IV. Both ATP-Pi exchange and Mg2(+)-ATPase activities were impaired by depletion of subunit IV from CF0-CF1. Partial restoration of these activities was obtained by reconstituting subunit IV-deficient CF0-CF1 with subunit IV. The impairment of these activities was likely caused by a loss in proton conductivity of CF0 upon removal of subunit IV. The dicyclohexylcarbodiimide-sensitive Mg2(+)-ATPase of subunit IV-deficient CF0-CF1 was not as sensitive to the depletion of subunit IV as ATP-Pi exchange. Nearly 90% of subunit IV could be removed, but Mg2(+)-ATPase activity was inhibited by only 40-60%. Thus subunit IV of CF0-CF1 may not participate directly in proton transfer but may have a role in organizing and/or stabilizing CF0 structure.
Feng
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
McCarty
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000502:physiology; D008081:Liposomes; D046911:Macromolecular Substances; D008564:Membrane Potentials; D010944:Plants; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000502:physiology; D013329:Structure-Activity Relationship
D008081:Liposomes; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases