Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria

1 Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X‐100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate‐binding site are located on the same side of the membrane. 2 The decarboxylase was solubilized with Triton X‐100 and purified by affinity chromatography on monomeric avidin‐Sepharose. The enzyme is composed of three types of polypeptides: the group of α chains ( M r 120000–140000) containing the biotin, the β chain (60000) and an apparently hydrophobic γ chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na + channel. The β and γ chains but not the α chain could be labelled by N ‐ethyl‐[ 14 C]maleimide. Similar decarboxylases but with much smaller biotin peptides ( M r 15000–20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum . 3 The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na + uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K + indicating an electrogenic Na + pump. The coupling between Na + transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. 4 The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl‐CoA decarboxylase are able to ferment glutamate and require 10 mM Na + for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.