The bilirubin-binding motif of bilitranslocase and its relation to conserved motifs in ancient biliproteins.
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.
Battiston
L
L
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
Passamonti
S
S
Macagno
A
A
Sottocasa
G L
GL
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
D000906:Antibodies; D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D013448:Sulfobromophthalein; D010798:Phycocyanin; D009525:Niacin; D002570:Ceruloplasmin; C033838:bilitranslocase; D001663:Bilirubin