title: Na+-pyrophosphatase: a novel primary sodium pump.
abstract: Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from the hyperthermophilic bacterium Thermotoga maritima catalyze Na+ rather than H+ transport into Escherichia coli inner membrane vesicles (IMV). When assayed in uncoupled IMV, the three PPases exhibit an absolute requirement for Na+ but display the highest hydrolyzing activity in the presence of both Na+ and K+. Steady-state kinetic analysis of PPi hydrolysis by Mm-PPase revealed two Na+ binding sites. One of these sites can also bind K+, resulting in a 10-fold increase in the affinity of the other site for Na+ and a 2-fold increase in maximal velocity. PPi-driven 22Na+ transport into IMV containing Mm-PPase was unaffected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, inhibited by the Na+ ionophore monensin, and activated by the K+ ionophore valinomycin. The Na+ transport was accompanied by the generation of a positive inside membrane potential as reported by Oxonol VI. These findings define Na+-dependent PPases as electrogenic Na+ pumps. Phylogenetic analysis suggests that ancient gene duplication preceded the split of Na+- and H+-PPases.
authors.affiliation: Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
authors.forename: Anssi M
authors.initials: AM
authors.lastname: Malinen
authors.forename: Georgiy A
authors.initials: GA
authors.lastname: Belogurov
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
mesh_terms: D001665:Binding Sites; D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D043564:Inorganic Pyrophosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D007555:Isoxazoles / Q000737:chemistry; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D017020:Methanosarcina / Q000201:enzymology / Q000378:metabolism; D008985:Monensin / Q000494:pharmacology; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology / Q000378:metabolism; D014634:Valinomycin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D007555:Isoxazoles; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D014634:Valinomycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C031184:oxonol VI; D008985:Monensin; D012964:Sodium; D043564:Inorganic Pyrophosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium