title: Oxalate:formate exchange. The basis for energy coupling in Oxalobacter.
abstract: In the Gram-negative anaerobe, Oxalobacter formigenes, the generation of metabolic energy depends on the transport and decarboxylation of oxalate. We have now used assays of reconstitution to study the movements of oxalate and to characterize the exchange of oxalate with formate, its immediate metabolic derivative. Membranes of O. formigenes were solubilized with octyl-beta-D-glucopyranoside in the presence of 20% glycerol and Escherichia coli phospholipid, and detergent extracts were reconstituted by detergent dilution. [14C]Oxalate was taken up by proteoliposomes loaded with unlabeled oxalate, but not by similarly loaded liposomes or by proteoliposomes containing sulfate in place of oxalate. Oxalate transport did not depend on the presence of sodium or potassium, nor was it affected by valinomycin (1 microM), nigericin (1 microM), or a proton conductor, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (5 microM) when potassium was at equal concentration on either side of the membrane. Such data suggest the presence of an overall neutral oxalate self-exchange, independent of common cations or anions. Kinetic analysis of the reaction in proteoliposomes gave a Michaelis constant (Kt) for oxalate transport of 0.24 mM and a maximal velocity (Vmax) of 99 mumol/min/mg of protein. A direct exchange of oxalate and formate was indicated by the observations that formate inhibited oxalate transport and that delayed addition of formate released [14C]oxalate accumulated during oxalate exchange. Moreover, [14C]formate was taken up by oxalate-loaded proteoliposomes (but not liposomes), and this heterologous reaction could be blocked by external oxalate. Further studies, using formate-loaded proteoliposomes, suggested that the heterologous exchange was electrogenic. Thus, for assays in which N-methylglucamine served as both internal and external cation, formate-loaded particles took up oxalate at a rate of 2.4 mumol/min/mg of protein. When external or internal N-methylglucamine was replaced by potassium in the presence of valinomycin, there was, respectively, a 7-fold stimulation or an 8-fold inhibition of oxalate accumulation, demonstrating that net negative charge moved in parallel with oxalate during the heterologous exchange. The work summarized here suggests the presence of an unusually rapid and electrogenic oxalate2-:formate1- antiport in membranes of O. formigenes. Since a proton is consumed during the intracellular decarboxylation that converts oxalate into formate plus CO2, antiport of oxalate and formate would play a central role in a biochemical cycle consisting of (a) oxalate influx, (b) oxalate decarboxylation, and (c) formate efflux.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: V
authors.initials: V
authors.lastname: Anantharam
authors.forename: M J
authors.initials: MJ
authors.lastname: Allison
authors.forename: P C
authors.initials: PC
authors.lastname: Maloney
mesh_terms: D001693:Biological Transport, Active; D004734:Energy Metabolism; D005561:Formates / Q000378:metabolism*; D006089:Gram-Negative Anaerobic Bacteria / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials; D010070:Oxalates / Q000378:metabolism*; D019815:Oxalic Acid; D011510:Proteolipids; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D005561:Formates; D010070:Oxalates; D011510:Proteolipids; C020485:proteoliposomes; C030544:formic acid; D014634:Valinomycin; D019815:Oxalic Acid