These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Active-site-directed photolabeling of the melibiose permease of Escherichia coli.
    Author: Ambroise Y, Leblanc G, Rousseau B.
    Journal: Biochemistry; 2000 Feb 15; 39(6):1338-45. PubMed ID: 10684614.
    Abstract:
    Covalent photolabeling of the melibiose permease (MelB) of Escherichia coli has been undertaken with the sugar analogue [(3)H]-p-azidophenyl alpha-D-galactopyranoside ([(3)H]-alpha-PAPG) with the purpose of identifying the domains forming the MelB sugar-binding site. We show that alpha-PAPG is a high-affinity substrate of MelB (K(d) = 1 x 10(-)(6) M). Its binding to or transport by MelB is Na-dependent and is competitively prevented by melibiose or by the high-affinity ligand p-nitrophenyl alpha-D-galactopyranoside (alpha-NPG). Membrane vesicles containing overexpressed histidine-tagged recombinant MelB were photolabeled in the presence of [(3)H]-alpha-PAPG by irradiation with UV light (lambda = 250 nm). Eighty-five percent of the radioactivity covalently associated with the vesicles was incorporated in a polypeptide corresponding to MelB monomer. MelB labeling was completely prevented by an excess of melibiose or alpha-NPG during the assay. Radioactivity analysis of CNBr cleavage or limited proteolysis products of the purified [(3)H]-alpha-PAPG-labeled transporter suggests that several domains of MelB are targets for labeling. One of the labeled CNBr cleavage products is a peptide with an apparent molecular mass of 5.5 kDa. It is shown that (i) its amino acid sequence is that of the Asp124-Met181 domain of MelB (7.5 kDa), which includes the cytoplasmic loop 4-5 connecting helices IV and V, the hydrophobic helix V, and the outer loop connecting helices V-VI, and (ii) that Arg141 in loop 4-5 is the only labeled amino acid of this peptide. Labeling of loop 4-5 provides independent evidence that this specific domain plays a significant role in MelB transport. Comparison with the well-characterized equivalent domain of LacY suggests that sugar transporters with similar structure and substrate specificity may have conserved domains involved in sugar recognition.
    [Abstract] [Full Text] [Related] [New Search]