The staphylococcal phosphoenolpyruvate-dependent phosphotransferase system. Purification and characterisation of the galactoside-specific membrane-component enzyme II

• Published in: European journal of biochemistry Vol. 113; no. 2; pp. 289 - 294
• Main Authors: Schäfer, A, Schrecker, O, Hengstenberg, W
• Format: Journal Article
• Language: English
• Published: England 01.01.1981
• Subjects: Amino Acids - analysis; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Galactosides - metabolism; Isoelectric Focusing; Lac Operon; Lipids - analysis; Membranes - enzymology; Molecular Weight; Phosphoenolpyruvate Sugar Phosphotransferase System - isolation & purification; Staphylococcus aureus - enzymology
• ISSN: 0014-2956
• DOI: 10.1111/j.1432-1033.1981.tb05065.x

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.