Purification of Escherichia coli B-specific p-aminobenzoate "pick-up" protein to homogeneity by affinity chromatography.
Of the satellite fractions of Escherichia coli B dihydrofolate synthetases, a non-enzymic protein that is specifically able to bind p-aminobenzoate and sulphonamides has been purified 6000-fold by p-aminobenzoylcellulose affinity chromatography. The protein was named p-aminobenzoate "pick-up" protein according to its function, i.e., to bring p-aminobenzoate into reaction with L-glutamate and pteridine during dihydrofolate biosynthesis. About 4 mg of pure protein (0.532% recovery, calculated from the total p-aminobenzoate binding capacity of the unfractionated supernatant separated from the crude bacterium plasma) can be obtained from 500 g of harvested cells. The product is homogeneous in polyacrylamide gel electrophoresis both in the absence and presence of sodium dodecyl sulphate, and has a molecular weight of 15,000 daltons +/- 5% as measured by sodium dodecyl sulphate gel electrophoresis and Sephadex G-75 gel column chromatography. p-Aminobenzoate and sulphonamide ligand binding studies showed a single binding site per p-aminobenzoate pick-up protein molecule. KD values for p-aminobenzoate and some sulphonamides as well as for L-glutamate, L-gamma-glutamyl oligopeptides, some pteridines and folate antagonists are also presented in order to illustrate the specificity of the receptor protein.[1]References
- Purification of Escherichia coli B-specific p-aminobenzoate "pick-up" protein to homogeneity by affinity chromatography. Toth-Martinez, B.L., Dinya, Z., Hernádi, F. J. Chromatogr. (1975) [Pubmed]
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