Purification and Characterization of a New Sodium‐Transport Decarboxylase

Abstract

Upon resolution of the particulate cell fraction of V. alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxyalse was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methlymalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles 8 times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km .apprxeq. 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km .apprxeq. 7 .mu.M), malonyl-CoA was also decarboxylated (apparent Km .apprxeq. 35 .mu.M). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO3- as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of 4 different polypeptides .alpha., .beta., .gamma., .delta., with MW 60,000, 33,000, 18,500 and 14,000. The latter of these polypeptides was clearly visible only after Ag-staining, but not after staining with Coomassie brilliant blue. A low MW polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin/125,500 g protein which was localized exclusively in the .gamma.-subunit. This subunit represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.