Characterization of the Membrane Protein Component of the Lactose Transport System of Escherichia coli

The membrane-localized lactose permease protein of Escherichia coli (M protein), which is the product of the y gene of the lac operon, was specifically labeled with radioactive N-ethylmaleimide. The labeled protein was effectively extracted from the particulate, membrane-containing fraction only in the presence of detergents, one of the most effective of which was sodium dodecyl sulfate. During electrophoresis on polyacrylamide gels in buffers containing sodium dodecyl sulfate, the labeled M protein ran as a single peak. Under these conditions, which promote the dissociation of many proteins into subunits, its molecular weight is about 30,000 as determined from its relative electrophoretic mobility. Chromatography of extracts of membrane proteins on Sephadex G-150 equilibrated with buffers containing sodium dodecyl sulfate was found to be a useful method of fractionation, which also leads to an effective separation of phospholipid from these proteins. Analysis on Sephadex G-150 confirms the value of about 30,000 for the molecular weight of the labeled M protein in buffer containing sodium dodecyl sulfate. The amount of M protein in cells grown on labeled amino acids, using gel electrophoresis to locate the protein, was estimated to be about twice that of thiogalactoside acetylase.