Purification and Characterization of a New Sodium‐Transport Decarboxylase

Abstract

Upon resolution of the particulate cell fraction of Veilionella aclcalescens by chromatography, membranes and ribosomes were celarly resolved. Methylmalony‐CoA decarboxylase was bound to the membranes and not to ribosomes as reported earler. Membrane vesicels containing mentylmalonyl‐CoA decarboxylase were prepared by disrupting V. alcalescens cells with French pressure chdmaber. About 64% of the decaroxylase was oriented in these vesicles with the sbstrated binding site facing to the outside. The vesicels perforemd a rapid acdumulation of Na+ ions in respon se to the decarboylatino of methylmalonyl‐CoA.Decarbosylation and transport wer highly uncouped. The effciency of the transport was considerably increased if methylmalonyl‐CoA cecarbosylation was retared by using a low temperature of by slowaly genrating the substrate enzymically form priopionyl‐CoA. Under optimized coditions Na+ was concentrated inside the inverted vesicles eight‐times than in the incuabaion medium. Mehtylmalonyl‐CoA decarboxylase was solubilized formt eh membranes with Tritom X‐100 and purified about 20‐fp;d nu affinity chromatography on monomeric avidin‐Sepharose coulmns. The decarboxylase was specifically activated by Na+ ions (apparend Km∼ 0.6 mM). Wherase (S)‐methylmalonyl‐CoA was the superior substrte. (apparent km∼ 7 μM). The decarboxylation of methylmalonyl‐CoA yielded CO2 and not HCO3− as th primary reaction product. Analysis of the prurified enzyme by dodecylsulfate gel electrophoresis inducated the presence of four differnet polypetides α, β, γ, δ with Mr 60 0000, 33 0000, 18 500 and 14000. The letter of these polypetides was elearly visible only after stiaing but not after staining with Coomassie brilliant blue. A low moleualr weight polypetide wit similar staaining properties also found in oxaloacetate decarbvoxylase Methylmalonyl‐CoA decarboxlase contained about 1 mol convalently bound biotin per 125 500 g protien which was localized exculsively in the γ‐subunit. This subunit therfore represents th biotin carboysl carier protein of methylmalonyl‐CoA decarboxylase. A new very senstivitymethod for the dtection of biotin containg proteins is described.

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

“…The sources of materials were the same as in [3]. Phosphatidylcholine type I I-S and type IV-S were purchased from Sigma, n-octyl glucoside was from Boehringer, Mannheim.…”

“…Methylmalonyl-CoA decarboxylase from Veillonella alcalescens was purified by affinity chromatography as described [3]. Lipids from Klehsiella aerogenes and V. alcalescens were isolated as described [I 21.…”

“…All four subunits a, p, y, 6 [3] were incorporated but the relative amounts of a and y chains were considerably reduced in the reconstituted membranes. This effect is probably due to partial dissociation of the enzyme during avidin-Sepharose affinity chromatography, leading to a non-physiological composition of subunits in the decarboxylase preparations [3]. It is reasonable that excess hydrophilic subunits are not incorporated into proteoliposomes (see Discussion).…”

“…In contrast to experiments with bacterial membrane vesicles [3], the Na" gradient did not decline rapidly after exhaustion of methylmalonyl-CoA, but Sodium uptake was determined in incubation mixtures containing the reconstituted decarboxylase (40 pg protein) and "NaCI (0.15 mM, 2600 counts min-' x nmol-') in a total volume of 0.65 ml 30 mM potassium phosphate buffer, pH 7.0. The uptake was initiated with 200 nmol (R,S)-methylmalonyl-CoA and measured as described [3] remained fairly constant over a period of 60 min, thus indicating a considerable tightness of the proteoliposomal membranes for Na' ions. The initial velocity of N a + uptake into the vesicles was dependent on the concentration of methylmalonylCoA and followed Michaelis-Menten kinetics (apparent K , z 1.5 pM at 0.25 mM NaCI).…”

“…Methylmalonyl-CoA decarboxylase activity was determined as described [3]. In some experiments (indicated in the text) the decarboxylase activity was determined by the more sensitive fluorometric assay described above.…”