The staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and characterisation of the...

The galactoside-specific membrane-bound component of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system, enzyme IIlac, was purified to homogeneity. The purification procedure involved several extractions steps at the particulate state, followed by solubilisation with Triton X-100. Up to this stage the biological activity of enzyme II was preserved. Isolation of the homogeneous protein involved gel filtration of the dodecylsulfate-denatured material. An apparent molecular weight of the polypeptide chain was estimated by dodecylsulfate gel electrophoresis. The 55000-Mr protein is visible in dodecylsulfate gels upon induction of the staphylococcal lac operon as a more intensively stained area. Antibodies against the denatured 55000-Mr protein inhibit the mutant complementation assay of enzyme II offered as membrane fragments. This demonstrates that the 55000-Mr protein and enzyme IIlac are identical. Polarity and the solubility of the protein in detergents are typical for an integral membrane protein.