PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF BETA-GALACTOSIDASE OF ESCHERICHIA COLI

1. 1. In constitutive or fully induced lactose fermenting strains of Escherichia coli, the β-galactosidase (EC 3.2.1.23) activity may be separated into at least ten well-defined zones of activity on polyacrylamide disc gels. A method is presented for the purification of these multiple forms of β-galactosidase exclusive of the major (95%) tetrameric species of this enzyme. 2. 2. Ultracentrifugation of a purified mixture of all isoenzymes yields six species with sedimentation values of approx. 16, 23, 27, 32, 36, and 41-45 S which are estimated to be aggregates of 4N, 6N, 8N, 10N, 12N, and 16N, respectively. 3. 3. The 16-S species has previously been shown to be a tetramer composed of identical subunits. The monomeric units of the 16-S and the 23-45-S species produced in urea appear to be identical by ultracentrifugation, electrophoresis in polyacrylamide disc gels, and amino acid composition. 4. 4. Mixtures of the 23-45-S species were found to be more sensitive than tetramer to thermal inactivation and denaturation. Dissociation of the tetramer in urea has previously been shown to proceed directly to monomer. Dissociation of the higher molecular weight forms proceeded through intermediate forms with sedimentation coefficients of approx. 16 and 10, presumably representing tetramer and dimer. 5. 5. Renaturation of urea-denatured heavy isoenzymes results in the formation of tetramers but not the heavier forms. This renatured tetramer is indistinguishable from the 16-S renatured tetramer in its sensitivity to urea and heat. 6. 6. The isoenzymes do not represent macromolecular complexes of β-galactosidase with other cell proteins or nucleic acids. © 1969.