Identification, Purification, and Partial Characterization of an Organic Anion Binding Protein from Rat Liver Cell Plasma Membrane

Allan W. Wolkoff; Cathie T. Chung

doi:10.1172/JCI109770

Identification, Purification, and Partial Characterization of an Organic Anion Binding Protein from Rat Liver Cell Plasma Membrane

Allan W. Wolkoff and Cathie T. Chung

Published May 1, 1980 - More info

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Abstract

Uptake of bilirubin, sulfobromophthalein (BSP), and other organic anions by the liver is a process with kinetics consistent with carrier mediation. The molecular basis of this transport mechanism is unknown. In the search for the putative organic anion carrier or receptor, the interaction of BSP with rat liver cell plasma membrane (LPM) has been studied. Specific binding of [³⁵S]BSP to LPM was determined using a filtration assay. Results revealed high affinity (K_a = 0.27 μM⁻¹), saturable (6.3 nmol/mg protein) binding, which was eliminated after preincubation with trypsin. Although [³⁵S]BSP was strongly bound to LPM, the binding was rapidly reversible, preventing direct identification and study of a specific binding site(s). To avoid this problem, a photoaffinity probe was devised, in which [³⁵S]BSP is covalently bound to LPM after exposure to ultraviolet light. Subsequent sodium dodecyl sulfate gel electrophoresis and fluorography revealed radioactivity predominantly associated with a single 55,000-mol wt protein. A protein with identical electrophoretic mobility was purified from deoxycholate solubilized LPM after affinity chromatography on glutathione-BSP-agarose gel. This protein migrated as a single band on sodium dodecyl sulfate gel electrophoresis and on urea gel isoelectric focusing. It contained 1-2 residues of sialic acid per 55,000-dalton protein, and was immunologically distinct from rat albumin and ligandin. It bound bilirubin with a K_d of 20 μM, as determined by tryptophan fluorescence quenching. Although the high affinity of this LPM protein for organic anions suggests that it may function as a hepatocellular organic anion receptor, its role in transport of these compounds is unknown.

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