title: Passive penetration of nitrate through the plasma membrane of Paracoccus denitrificans and its potentiation by the lipophilic tetraphenylphosphonium cation.
abstract: Previously, it has been shown that treatment of Paracoccus denitrificans cells with phenylglyoxal inhibits the methyl-viologen-linked nitrate reductase activity by blocking the nitrate transporter. This inhibition disappears if tetraphenylphosphonium cation (TPP(+)) is added to the assay medium. In the present paper, the following evidence suggests that the effect of TPP(+) results from an increased transmembrane anion permeability and not from transporter reactivation or cell lysis. (1) Beside nitrate, TPP(+) also mediated the utilisation of chlorate, which normally lacks access to the cytoplasm. (2) The TPP(+) pathway had about hundred-times higher K(m) values for nitrate and chlorate than nitrate reductase in Triton X-100 permeabilised cells. (3) Although the uncoupler CCCP alone failed to overcome the PG block, it stimulated the operation of the TPP(+) pathway. (4) The method of continuous variations allowed the transport stoichiometry TPP(+)/NO(3)(-) to be determined as 3, indicating charge compensation for nitrate movement and the subsequent transmembrane two-electron redox reaction. Anion uptake was also measured independently from passive swelling of uncoupled spheroplasts in iso-osmotic solutions of ammonium salts. The permeability to nitrate lay in the permeability sequence Cl(-)<NO(3)(-)<ClO(4)(-)<SCN(-) and was further enhanced by TPP(+).
authors.affiliation: Department of Biochemistry, Faculty of Science, Masaryk University, Kotlárská 2, CZ-61137 Brno, Czech Republic. ikucera@chemi.muni.cz
authors.forename: Igor
authors.initials: I
authors.lastname: Kucera
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002704:Chlorates / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D009566:Nitrates / Q000737:chemistry / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010231:Paracoccus denitrificans / Q000378:metabolism*
chemical_list: D002704:Chlorates; D009566:Nitrates; D009861:Onium Compounds; D009943:Organophosphorus Compounds; C013289:tetraphenylphosphonium