Reconstitution of Na+ transport from purified methylmalonyl-CoA decarboxylase and phospholipid vesicles.
Methylmalonyl-CoA decarboxylase from Veillonella alcalescens which was isolated by affinity chromatography on avidin-Sepharose was incorporated into phospholipid vesicles by the detergent dilution method with octyl glucoside as the detergent. By this procedure the Na+ pump activity was reconstituted. The optimal octyl glucoside concentration for reconstitution was about 2.8%. The activity of reconstituted Na+ transport increased with the amount of enzyme present during reconstitution until a plateau was reached at about 7 micrograms enzyme/mg phospholipid. All four polypeptides of the decarboxylase were incorporated into the proteoliposomes but the relative amounts of alpha and gamma subunits were considerably reduced. The reconstitution process was highly asymmetric, since about 80% of the decarboxylase was oriented in the proteoliposomes with the substrate binding site facing to the interior. The orientation was determined from the increase of methylmalonyl-CoA decarboxylase activity by destruction of the membrane permeability barrier. It was also deduced from the amount of enzyme which was not accessible from the outside to inactivation by avidin. With these reconstituted vesicles, a steady-state internal Na+ concentration was established by methylmalonyl-CoA decarboxylation which under optimized conditions was about 30-fold higher than in the incubation medium. Sodium ion accumulation in the presence of the Na+-carrying ionophores nigericin or monensin was practically nil. In the presence of valinomycin or carbonylcyanide-p-trifluoromethoxy phenylhydrazone the rate of Na+ transport and its steady-state internal concentration were considerably higher than in the controls which is consistent with the function of methylmalonyl-CoA decarboxylase as an electrogenic Na+ pump.
D001692:Biological Transport; D002262:Carboxy-Lyases / Q000378:metabolism*; D002846:Chromatography, Affinity; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D043586:Methylmalonyl-CoA Decarboxylase; D010743:Phospholipids / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D014678:Veillonella / Q000201:enzymology*
D008081:Liposomes; D010743:Phospholipids; D012964:Sodium; D002262:Carboxy-Lyases; D043586:Methylmalonyl-CoA Decarboxylase