Purification by affinity chromatography of a membrane dicarboxylate binding protein from Bacillus subtilis

Membrane vesicles of Bacillus subtilis W23 actively transport the C4 and C5 dicarboxylates of the tricarboxylate cycle by system(s) of relatively high affinity for their requisite substrates (Km 4-53 microM). Glutamate and succinate binding activities were readily solubilized from membrane vesicles by nonionic detergents, particularly by Lubrol WX. From this extract, glutamate binding activity was highly enriched by affinity chromatography on phloroglucinol-expanded Sepharose-6B to which L-aspartate was coupled via divinylsulfone. Another protein (41000 molecular weight), which bound both L-glutamate and L-malate, was purified from affinity columns to which either L-glutamate or L-malate had been coupled via bisdiglycidyl ether. This protein bound labelled L-malate as well as L-glutamate with affinities similar to those seen with membrane vesicles (Kd's 8 microM L-malate and 52 microM L-glutamate).