Abe K, Hayashi H, Maloney P C
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
J Biol Chem. 1996 Feb 9;271(6):3079-84. doi: 10.1074/jbc.271.6.3079.
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.
我们通过重组来监测载体（称为AspT）的活性，研究了乳酸杆菌亚种M3中天冬氨酸代谢被组织成质子动力代谢循环的这一观点。该载体预期会进行前体（天冬氨酸）和产物（丙氨酸）的电生成交换。在0.4%大肠杆菌磷脂和20%甘油存在的情况下，用1.25%辛基葡糖苷提取乳酸杆菌亚种M3的膜。然后将提取物用于制备负载有天冬氨酸或丙氨酸的蛋白脂质体。负载天冬氨酸的蛋白脂质体通过与内部底物交换积累外部的[3H]天冬氨酸；这种同源自交换（Kt = 0.4 mM）对钾或质子离子载体不敏感，并且不受Na+、K+或Mg2+存在与否的影响。负载丙氨酸的蛋白脂质体也在异源反向转运反应中摄取[3H]天冬氨酸，该反应分别受到膜电位内正或内负的刺激或抑制。几条证据表明这些同源和异源交换反应是由相同的功能单元催化的。因此，在自交换过程中被AspT摄取的[3H]天冬氨酸会因延迟添加丙氨酸而释放。此外，在重组前将去污剂提取物在37℃保存时发生的AspT活性的自发丧失，会因天冬氨酸（KD（天冬氨酸） = 0.3 mM）或丙氨酸（KD（丙氨酸）≥10 mM）的存在而被阻止，这表明两种底物都直接与AspT相互作用。这些发现与乳酸杆菌亚种M3在天冬氨酸代谢过程中质子动力代谢循环的运作是一致的。
