TY - JOUR
T1 - Identification, purification, and partial characterization of an organic anion binding protein from rat liver cell plasma membrane
AU - Wolkoff, A. W.
AU - Chung, C. T.
PY - 1980
Y1 - 1980
N2 - 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.
AB - 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.
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U2 - 10.1172/JCI109770
DO - 10.1172/JCI109770
M3 - Article
C2 - 7364942
AN - SCOPUS:0018968775
SN - 0021-9738
VL - 65
SP - 1152
EP - 1161
JO - Journal of Clinical Investigation
JF - Journal of Clinical Investigation
IS - 5
ER -