Abstract
Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.
Highlights
Gel filtration of male rat liver cytosol preincubated effects asfree bile acids
The cytosolic glutathione S-transferwith radiolabeled lithocholic,chenodeoxycholic, and ases have been viewed as the major intracellular binders of glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the otherwith a separatefraction, respectively
Purification of organic anion binder (D,.w)as as previously described (9).Glutathione S-transferasesA, B, and Cwere purified from rat liver according to Habig et al (11)and found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be homogeneous with the expected subunit compositions Y h Y h, Y,Y, YhYt, respectively
Summary
From the GastroenterologySection, Medical and Research Services, Wadsworth Veterans Administration Hospital Center a n d UCLA School of Medicine, Los Angeles, California, 90073. Bialecid binding to purified Binders I and I1 and glutathione S-transferases A, B, and C was both bile acids and organic anions (1-7). We have identified and isolated two proteins (Binders I and 11) which, along with glutathione S-transferase B, are themajor hepatic cytosol bile acid binding proteins; these pro-. Pooled fractions containing the glutathione S-transferases or the by the paymentof page charges.This article must be herebnyew bile acid binding activity were prepared by repetitive gel filtration marked “advertisement” in accordance with 18 U.S.C. Section 1734 (X3) as previously described (9). Aliquots (0.5 ml) of individual column fractions from gel filtration of hepatic cytosol were dialyzed for 40 h against 1 liter of Buffer A containing radioactive lithocholic or chenodeoxycholic acid (76 and 5.5 nM, respectively). Radioactivity on both sides of the membrane were determined andexpressed as Ch/C,, where Cb is the bound concentration determined by subtracting the free concentration (C,) on the protein-free side from the total concentration in the protein-containing side
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