A bifunctional enzyme aggregate catalyzes the first two steps of tryptophan biosynthesis in Salmonella typhimurium. The complex is composed of an arrangement of two nonidentical protein components, the anthranilate synthetase subunit and the 5-phosphorylribose 1-pyrophosphate phosphoribosyl transferase subunit. A method of purification of the complex has been devised which makes use of two properties of the system: (1) when mixed in vitro, the uncomplexed subunit proteins undergo a rapid and spontaneous selfassembly forming the active complex, and (2) auxotrophic mutant strains of 5. typhimurium are available which lack either the anthranilate synthetase component or the 5-phosphorylribose 1-pyrophosphate phosphoribosyl transferase component and thus can serve as sources of uncomplexed 5-phosphorylribose 1-pyrophosphate phosphoribosyl transferase and anthranilate synthetase subunits, respectively. Partially purified extracts of two such mutant strains were subjected to preliminary, parallel fractionation by Sephadex G-100 gel filtration. These two subunit preparations were subsequently combined permitting spontaneous aggregation of the subunits into the complex. The pooled preparation was then refractionated by repeating the Sephadex G-100 gel filtration. The newly formed anthranilate synthetase-5-phosphorylribose 1-pyrophosphate phosphoribosyl transferase complex emerged from the column characteristically in the excluded volume preceding the residual uncomplexed subunits and the other nonspecific components which were present in the mixture. A high degree of purity of the isolated complex was indicated by ultracentrifugational analysis and disc gel electrophoresis. This technique of “complementation-coupled gel filtration” might readily be applied to the purification of other enzyme or protein aggregates. © 1969, American Chemical Society. All rights reserved.
