EXCHANGE OF ASPARTATE AND ALANINE - MECHANISM FOR DEVELOPMENT OF A PROTON-MOTIVE FORCE IN BACTERIA

We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstit ution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and prod uct (alanine), Membranes of Lactobacillus subsp. M3 were extracted wit h 1.25% octyl glucoside in the presence of 0.4% Escherichia coli phosp holipid and 20% glycerol. The extracts were then used to prepare prote oliposomes loaded with either aspartate or alanine. Aspartate-loaded p roteoliposomes accumulated external [H-3]aspartate by exchange with in ternal substrate; this homologous self-exchange (K-t = 0.4 mM) was ins ensitive to potassium or proton ionophores and was unaffected by the p resence or absence of Na+, K+, or Mg2+. Alanine-loaded proteoliposomes also took up [H-3]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative m embrane potential, respectively, Several lines of evidence suggest tha t these homologous and heterologous exchange reactions were catalyzed by the same functional unit, Thus, [H-3]aspartate taken up by AspT dur ing self-exchange was released by a delayed addition of alanine, In ad dition, the spontaneous loss of AspT activity that occurs when a deter gent extract is held at 37 degrees C prior to reconstitution was preve nted by the presence of either aspartate (K-D(aspartate) = 0.3 mM) or alanine (K-D(alanine) greater than or equal to 10 mM), indicating that both substrates interact directly with AspT, These findings are consi stent with operation of a proton motive metabolic cycle during asparta te metabolism by Lactobacillus subsp. M3.