title: The bilirubin-binding motif of bilitranslocase and its relation to conserved motifs in ancient biliproteins.
abstract: In the primary structure of bilitranslocase, currently under study in our laboratory, an aminoacid motif was identified and found to be conserved in a number of alpha-phycocyanines, ancient biliproteins present in cyanobacteria. To test the possibility that such a motif could be at least part of the binding site for bilirubin, epitope-specific antibodies were raised. The target corresponds to the sequence 65-75 of bilitranslocase and covers the central portion of the motif identified. The antibodies were shown: 1) to inhibit the electrogenic BSP transport by plasmamembrane vesicles; 2) to react with purified bilitranslocase; and 3) to identify only one protein band with electrophoretic mobility identical to bilitranslocase in Western blots of solubilised plasmamembrane vesicles. The presence of either bilirubin or nicotinate during pre-incubation with the antibodies decreases concentration-wise the inhibition kinetics. From these experiments a dissociation constant of 2.2 +/- 0.3 and 11.3 +/- 1.3 nM for bilirubin-bilitranslocase and nicotinate-bilitranslocase complexes were calculated.
authors.affiliation: Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Battiston
authors.forename: S
authors.initials: S
authors.lastname: Passamonti
authors.forename: A
authors.initials: A
authors.lastname: Macagno
authors.forename: G L
authors.initials: GL
authors.lastname: Sottocasa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001663:Bilirubin / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002570:Ceruloplasmin; D017124:Conserved Sequence / Q000235:genetics; D000939:Epitopes / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000502:physiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009525:Niacin / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D010798:Phycocyanin / Q000031:analogs & derivatives / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013448:Sulfobromophthalein / Q000378:metabolism
chemical_list: D000906:Antibodies; D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D013448:Sulfobromophthalein; D010798:Phycocyanin; D009525:Niacin; D002570:Ceruloplasmin; C033838:bilitranslocase; D001663:Bilirubin