title: Limited proteolysis of lactose permease from Escherichia coli.
abstract: Escherichia coli lactose permease (also referred to as lactose carrier) is an integral protein of the cytoplasmic membrane. Using lactose permease either radiolabeled biosynthetically in plasmid-bearing E. coli minicells or radioalkylated post-synthetically by chemical modification, we have determined sites on the membrane-bound protein accessible to proteolytic attack and we have characterized several high-molecular-mass products. The most prominent polypeptide obtained from lactose permease radiolabeled biosynthetically is observed after digestion with different proteases. The fragment produced by thermolysin was shown to contain the intact N-terminus and to extend into the region around amino acid residue 140 which, according to secondary structure models, is presumed to be less tightly folded than the rest of the molecule. Evidence is presented that the corresponding fragments obtained after digestion with several other proteases also originate from the N-terminal part of the protein. This N-terminal segment of the lactose carrier is resistant to proteolytic digestion even in the presence of non-ionic detergents and it may represent a tightly folded domain. Additional proteolytic cleavage sites located C-terminal of the Cys148 residue can be inferred.
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000378:metabolism; D006868:Hydrolysis; D026901:Membrane Transport Proteins / Q000032:analysis*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins; D017830:Octoxynol; D010447:Peptide Hydrolases; D011092:Polyethylene Glycols / Q000494:pharmacology; D027981:Symporters
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D011092:Polyethylene Glycols; D017830:Octoxynol; C021499:lactose permease; D010447:Peptide Hydrolases; D003545:Cysteine; D005033:Ethylmaleimide