title: The proteolipid subunit of the chloroplast adenosine triphosphatase complex. Reconstitution and demonstration of proton-conductive properties.
abstract: The dicyclohexylcarbodiimide (DCCD)-binding proteolipid of the chloroplast ATPase complex was solubilized in 1-butanol, isolated by ion exchange chromatography, and reconstituted in a liposomal system. Proton-conducting activity was monitored by fluorometry with 9-aminoacridine as an indicator of delta pH in K+-loaded liposomes suspended in a K+-free medium. Addition of valinomycin served to create a membrane potential. Proton mediation was further followed potentiometrically with a pH electrode. Reconstituted chloroplast DCCD-binding proteolipid rapidly catalyzed passive proton movement as measured by the quenching of 9-aminoacridine fluorescence upon addition of valinomycin to K+-loaded vesicles. Proton translocation was inhibited to approximately 80% by hydrophobic dicyclohexylcarbodiimide but not its water-soluble analog (1-ethyl-3[3-dimethylaminopropyl]carbodiimide). Maximal inhibition by DCCD occurred after 60 min of incubation with 20 to 40 nmol of DCCD/20 nmol of proteolipid/10 mumol of lipid. The velocity of proton conduction increased when the external proton concentration in the medium was increased. Similar data were obtained using a pH electrode to follow proton movement. Subjection of proteolipid liposomes to tetranitromethane, resulting in nitration of tyrosine, markedly reduced proton conduction (62% inhibition). In a similar fashion preincubation of reconstituted vesicles with hydrophobic phenylisothiocyanate at neutral pH resulted in a decreased rate of proton movement (50% inhibition) while hydrophilic p-sulfophenylisothiocyanate appeared to have no effect.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002736:Chloroplasts / Q000187:drug effects / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D006017:Glycolipids / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D010944:Plants / Q000378:metabolism; D011199:Potentiometry; D011510:Proteolipids / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D006017:Glycolipids; D008081:Liposomes; D046911:Macromolecular Substances; D011510:Proteolipids; D014634:Valinomycin; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases