Identification, purification, and partial characterization of an organic anion binding protein from rat liver cell plasma membrane

A. W. Wolkoff, C. T. Chung

Research output: Contribution to journalArticlepeer-review

98 Scopus citations

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 (Ka+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 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 Kd 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.

Original languageEnglish (US)
Pages (from-to)1152-1161
Number of pages10
JournalJournal of Clinical Investigation
Volume65
Issue number5
DOIs
StatePublished - 1980

ASJC Scopus subject areas

  • Medicine(all)

Fingerprint Dive into the research topics of 'Identification, purification, and partial characterization of an organic anion binding protein from rat liver cell plasma membrane'. Together they form a unique fingerprint.

Cite this