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 reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0. 4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [3H]aspartate by exchange with internal substrate; this homologous self-exchange (Kt = 0.4 mm) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na+, K+, or Mg2+. Alanine-loaded proteoliposomes also took up [3H]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative membrane potential, respectively. Several lines of evidence suggest that these homologous and heterologous exchange reactions were catalyzed by the same functional unit. Thus, [3H]aspartate taken up by AspT during self-exchange was released by a delayed addition of alanine. In addition, the spontaneous loss of AspT activity that occurs when a detergent extract is held at 37 degrees C prior to reconstitution was prevented by the presence of either aspartate (KD(aspartate) = 0.3 mm) or alanine (KD(alanine) > or = 10 mm), indicating that both substrates interact directly with AspT. These findings are consistent with operation of a proton-motive metabolic cycle during aspartate metabolism by Lactobacillus subsp. M3.
Abe
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K
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Hayashi
H
H
Maloney
P C
PC
Malone
P C
PC
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D007778:Lactobacillus / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D008954:Models, Biological; D010743:Phospholipids / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D018892:Proton-Motive Force; D011522:Protons; D014634:Valinomycin / Q000494:pharmacology
D010743:Phospholipids; D011522:Protons; D014634:Valinomycin; D001224:Aspartic Acid; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D000409:Alanine; D005990:Glycerol; D011188:Potassium