Reconstitution of Na⁺ transport from purified methylmalonyl‐CoA decarboxylase and phospholipid vesicles

Abstract

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 7:. The activity of reconstituted N a + transport increased with the amount of enzyme present during reconstitution until a plateau was reached at about 7 pg enzyme/mg phospholipid. All four polypeptides of the decarboxylase were incorporated into the proteoliposomes but the relative amounts of a and y 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 methylmalonylCoA 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.

Sodium ion transport decarboxylases and other aspects of sodium ion cycling in bacteria.

“…Furthermore, tryptic hydrolysis of methylmalonyl-CoA decarboxylase yielded a number of small polypeptides but no specific cleavage to one with the molecular mass of the y-chain (unpublished results). Finally, upon reconstitution of methylmalonyl-CoA decarboxylase, all four subunits including the y-chain were incorporated into liposomes (81). From these observations it appears unlikely that the small subunit is an accidental contaminant, but final proof for its being a part of the decarboxylase probably requires elucidation of its function.…”

“…(ii) Evidence for ATP-driven Na+ and K+ transport has been obtained, e.g., in Streptococcias fJaecalis (77,89), Propionigenium inodlestiuon (83), and Esciherichia(coli (54,55,214). (iii) We have detected the Na'pumping oxaloacetate decarboxylase of Klebsie/lla Pln(eI noiiiae (38,39,41,45,46) and Salmonella tvplhimuriumn (45) and methylmalonyl-coenzyme A (CoA) decarboxylase of Veillone/ll/a a1(cI/aSCees (79)(80)(81)(82) and P. mlondestimtn (83) that pcrform Na+ extrusion at the expense of the free energy of decarboxylation reactions. Related Na t -pUnmpin-decarbox-ylases are the glutaconyl-CoA decarboxylases of Acidam-iflOC'OC'(Ul' teSiile tis, Pep tostrepto(cOc(cls asacc/Iaro/Yticus, and Clostriilidiami svmbiosum (20.…”

Carboxylation of pyruvate and acetyl coenzyme A by reversal of the sodium pumps oxaloacetate decarboxylase and methylmalonyl-CoA decarboxylase

“…Reconstitution of the 7Va+ Pumps Oxaloacetate Decarboxylase and Methylmalonyl-CoA Decarboxylase. The reconstitutions were performed as described (Dimroth, 1981; Hilpert & Dimroth, 1984) modified as follows: lipids from Klebsiella aerogenes (80 mg) were vigorously agitated on a Vortex mixer for about 10 min under nitrogen with 2.67 mL of 3% octyl glucoside in reconstitution buffer (30 mM potassium phosphate, pH 7.0, containing 1 mM Na2S04,0.5 mM dithioerythritol, and 1.5 mM NaN3). Depending on the experiments to be performed, oxaloacetate decarboxylase (0.08 mL, 0.35 mg, 20 units) and (or) methylmalonyl-CoA decarboxylase (0.05 mL, 0.26 mg, 7 units) were added to the lipid detergent mixture followed by dialysis against 1 L of reconstitution buffer for 3 days at 4 °C with two changes of the dialysis buffer.…”

“…This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. pumps were reconstituted in use of the same reconstitution procedure (Hilpert & Dimroth, 1984;Buckel & Semmler, 1983).…”

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus

“…The decarboxylase is bound to the cell membrane and is specifically activated by Na ϩ ions (13,14). The most important role of the enzyme is as a vectorial catalyst converting part of the energy of the highly exergonic decarboxylation reaction into an Na ϩ ion gradient.…”