Purification of bile acid-binding proteins from rat hepatic cytosol. Use of a photoaffinity label to detect novel Y' binders.
A 125I-labelled photolabile derivative of cholic acid has been used to investigate the organic anion-binding Y' fraction from rat liver, prepared by the method of Sugiyama, Y., Yamada, T. and Kaplowitz, N. (1982) Biochimica Biophysica Acta, 709, 342-352. The use of this photoaffinity probe led to the discovery of previously undescribed bile acid-binding proteins. A comprehensive purification scheme for the Y' proteins which allows the isolation of these novel binding species is described. Electrophoretic analysis shows that the Y' binders can be divided into two groups. The proteins in group 1 are dimeric and the 5B, 6E and 7F binding species consist of subunits with approximate molecular masses of 19.6, 15.6 and 14.9 kDa, respectively. The group 2 binding proteins, 5C, 5D and 8C, are monomeric and have molecular masses of approximately 36.2, 36.2 and 33 kDa, respectively. Calculation of the incorporation of 125I by these proteins showed that the group 1 proteins displayed a significantly greater specific incorporation of radioactivity than group 2. The specificity of 125I-labelled 3 beta-azidocholylhistamine is further demonstrated by analysis of tryptic digests of photoaffinity labelled Y' binders and glutathione S-transferases AA, A, D and F by reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The majority of the radioactivity was shown to be incorporated into a single component, which was not coincident with the free photoaffinity label.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006913:Hydroxysteroid Dehydrogenases; D008099:Liver / Q000166:cytology*; D008297:Male; D008562:Membrane Glycoproteins; D010777:Photochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D000345:Affinity Labels; D002352:Carrier Proteins; D005982:Glutathione Transferase; D006913:Hydroxysteroid Dehydrogenases; D008562:Membrane Glycoproteins; C037643:bile acid binding proteins; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)