title: Reconstituted Cl- pump protein: a novel ion(Cl-)-motive ATPase.
abstract: Cl- absorption by the Aplysia californica foregut is effected through an active Cl- transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl-, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl- accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl- accumulation and electrical potential while an outwardly-directed valinomycin-induced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl- accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate or p-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl- accumulation, intraliposomal negative potential difference, and also Cl(-)-stimulated ATPase activity. Both aspects of Cl- pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl(-)-ATPase actively transports Cl- by an electrogenic process.
authors.affiliation: Department of Physiology, College of Medicine, University of Florida, Gainesville 32610, USA.
authors.forename: G A
authors.initials: GA
authors.lastname: Gerencser
authors.forename: K R
authors.initials: KR
authors.lastname: Purushotham
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D027321:Anion Transport Proteins; D001048:Aplysia; D001639:Bicarbonates / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials; D011188:Potassium / Q000378:metabolism; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D027321:Anion Transport Proteins; D001639:Bicarbonates; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D008345:Manganese; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C031652:anion-sensitive ATPases; D008274:Magnesium; D011188:Potassium; D004229:Dithiothreitol