Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.
Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes, but not on other cell populations from normal rat liver. It was not found in other organs. These data are compatible with the hypothesis that a specific liver cell plasma membrane protein mediates the hepatocytic sequestration of bilirubin and BSP.
D000818:Animals; D001663:Bilirubin / Q000378:metabolism; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification*; D002462:Cell Membrane / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006651:Histocytochemistry; D005779:Immunodiffusion; D008099:Liver / Q000032:analysis*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013448:Sulfobromophthalein / Q000378:metabolism; D014018:Tissue Distribution
D002352:Carrier Proteins; D000939:Epitopes; C488927:bilirubin binding protein, rat; D013448:Sulfobromophthalein; D001663:Bilirubin