The generation of an electrochemical gradient of sodium ions upon decarboxylation of oxaloacetate by the membrane-bound and Na+-activated oxaloacetate decarboxylase from Klebsiella aerogenes.

Abstract

Inverted membrane vesicles from Klebsiella arragenes containing the Na+‐activated oxaloacctate decarboxylase accumulated 22Na+ in response to the decarboxylation of oxaloacetate. Complexation of the biotin prosthetic group of the decarboxylase with avidin completely prevented the oxaloacetate‐dependent Na+ uptake, whereas avidin saturated with biotin was without effect.The rapid accumulation of 22Na+ inside the vesicles following oxaloacetate addition was succeeded by a slower efflux of 22Na+ after exhaustion of oxaloacetate. Other substrates, e.g. d‐lactate, were unable to replace oxaloacetate in accumulating 22Na+ to a comparable extent, with the exception of (S)‐malate, which acted through its conversion to oxaloacetate. The amount of Na+ which was transported into the vesicles by a limited amount of oxaloacetate critically depended on the rate of oxaloacetate decarboxylation. This rate was retarded by using a low temperature (0 °C), by inhibiting the enzyme with oxalatc or by using oxaloacetate‐generating systems, where the activity of the enzyme catalyzing oxaloacetate formation was rate‐determining. With these retarded systems of oxaloacetate decarboxylation the efficiency of the Na+ transport was significantly increased. Under optimized conditions Na+could be concentrated inside the inverted vesicles eight‐times higher than in the incubation medium.The Na+‐carrying ionophores nigericin and trinactin effectively prevented the oxaloacetate‐dependent Na+ accumulation in a K+‐free incubation medium. In the presence of valinomycin and K+, however, the amount of Na+ which was taken up by the vesicles following oxaloacetate addition increased by a factor of two, which indicated limitation of Na+ uptake by electrical parameters. The generation of a membrane potential was determined from the distribution of the lipophilic anion [14C]SCN‐between interior and exterior vesicular spaces. The oxaloacetate‐decarboxylase‐driven Na+ transport into inverted vesicles at 0°C generated a membrane potential of 65 mV, an Na+ gradient equivalent to 49 mV and a total driving force of 114 mV. The proton carrier carbonylcyanide‐p‐trifluoromethoxy phenylhydrazone had essentially no effect on the oxaloacetate‐de‐carboxylase‐driven Na+ uptake, indicating that thc gcneration of a proton gradicnt is not involved. It is concluded from these results that oxaloacetate decarboxylase acts as a primary active Na+ pump which generates an electrochemical gradient of Na+ ions.