title: Characterization of an ATP-driven H+ pump in human placental brush-border membrane vesicles.
abstract: The presence of an ATP-driven H+ pump as measured by H+ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H+ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H+ pump from an inward-facing configuration to outward-facing. Consequently, H+ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H+ pump. The reoriented H+ pump was electrogenic because H+ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H+ uptake with an apparent Km of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg2+ and Mn2+ were the most preferred bivalent cations. Co2+ and Zn2+ showed partial activity, whereas Ca2+ and Ba2+ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A1 and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN3. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H+ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H+ pumps.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
authors.forename: B J
authors.initials: BJ
authors.lastname: Simon
authors.forename: P
authors.initials: P
authors.lastname: Kulanthaivel
authors.forename: G
authors.initials: G
authors.lastname: Burckhardt
authors.forename: S
authors.initials: S
authors.lastname: Ramamoorthy
authors.forename: F H
authors.initials: FH
authors.lastname: Leibach
authors.forename: V
authors.initials: V
authors.lastname: Ganapathy
mesh_terms: D000165:Acridine Orange; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001693:Biological Transport, Active; D002413:Cations, Divalent / Q000494:pharmacology; D002793:Cholic Acids / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018942:Macrolides; D008871:Microvilli / Q000378:metabolism / Q000502:physiology; D009711:Nucleotides / Q000494:pharmacology; D010920:Placenta / Q000378:metabolism* / Q000648:ultrastructure; D011247:Pregnancy; D017494:Proton Pumps / Q000187:drug effects / Q000502:physiology*; D012680:Sensitivity and Specificity; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D002413:Cations, Divalent; D002793:Cholic Acids; D018942:Macrolides; D009711:Nucleotides; D017494:Proton Pumps; D014634:Valinomycin; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000165:Acridine Orange; D005033:Ethylmaleimide