Isolation of two cholic acid-binding proteins from rat liver cytosol using affinity chromatography

Using an affinity matrix coupled with cholic acid, two proteins that recognise bile acids were isolated from rat liver cytosol. One protein of molecular weight 68 000 was immunologically identical to rat albumin. The other protein was of molecular weight 46 000. On discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis the 46 000 molecular weight protein dissociated to a single band with an RF value identical to the Yb subunit of the bromosulphophthalein-binding fraction (Y-fraction) of whole liver cytosol. The monomers of purified ligandin under these conditions resolved into two bands which corresponded to the Ya and Yc subunits of liver cytosol Y-fraction. Anti-serum to the purified ligandin reacted monospecifically with purified ligandin and whole liver cytosol, but did not cross-react with the Yb dimer eluted from the affinity column. The Yb dimer was shown to possess glutathione-S-transferase activity with a substrate specificity distinct from ligandin but similar to glutathione-S-transferase C. Cholic acid inhibited the catalytic activity of the transferase.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Haem-binding proteins of the rat liver cytosol

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino [³h] laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [³H] haem-labelled mitochondria, [³H] haem-labelled microsomes or with [³H] haemin.These results are discussed with particular reference to ligandin.     

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.     

Haem-binding proteins of the rat liver cytosol.

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino[3H]laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [3H]haem-labelled mitochondria, [3H]haem-labelled microsomes or with [3H]haemin. These results are discussed with particular reference to ligandin.     

Regulation of the biosynthesis of two distinct fatty acid-binding proteins in rat liver and intestine. Influences of sex difference and of clofibrate

Two distinct fatty acid-binding proteins (FABPs) have been identified in rat intestine, gFABP (15,063 Da) which is confined to intestinal epithelium and hFABP (14,184 Da) which is found in both liver and intestine. We have examined the influence of sex difference and the effect of clofibrate, both of which affect cellular fatty acid metabolism and hFABP levels, on the concentration, and mRNA levels of both hepatic and intestinal FABPs. In the liver, hFABP concentration was approximately 2-fold greater in females and in clofibrate-treated males than in untreated male rats. These differences were not accompanied by changes in the fractional turnover of the polypeptide but rather by parallel increases in hFABP mRNA. In the intestine, the two FABPs exhibited different regulatory responses. Intestinal hFABP turnover was 33% greater in females than in males, whereas mRNA concentration was 50% greater. Thus, unlike hFABP in liver, there was no sex-related difference in the steady-state level of hFABP in intestine. However, clofibrate treatment, similar to its effects in the liver, doubled intestinal hFABP protein and mRNA concentration. In contrast to hFABP, neither gFABP protein nor mRNA concentration were sex dependent, whereas clofibrate produced only a modest increase in gFABP concentration without significantly changing gFABP mRNA levels. The results indicate that the influence of sex difference and the effect of clofibrate on hepatic fatty acid metabolism are both associated with changes in hFABP synthesis mediated pretranslationally. The differential response of hFABP and gFABP in intestine suggests that these proteins play distinct roles in the cellular metabolism of fatty acids.     

Partial purification of two lithocholic acid-binding proteins from rat liver 100 000g supernatants.

1. The partial purification of two lithocholic acid-binding proteins from liver 100 000g supernatants is described. 2. Gel-filtration, (NH4)2SO4 fractionation, Ca3(PO4)2 fractionation and ion-exchange chromatography were used. 3. Both proteins exhibited glutathione S-transferase activity; one may be the non-specific anion-binding protein ligandin. 4. Glutathione S-transferase activity of one of the binding proteins was inhibited by lithocholic acid.     

Studies on fatty acid-binding proteins. The binding properties of rat liver fatty acid-binding protein.

The inactive 2Fe species of the Fe protein of the nitrogenase of Klebsiella pneumoniae was generated by treating oxidized Fe protein (Kp2) with MgATP and chelator. Incubation of the 2Fe species of Kp2 with the sulphurtransferase rhodanese in the presence of thiosulphate, ferric citrate and reduced lipoate reproducibly restored activity. The extent of restoration of activity depended on the molar ratio of 2Fe Kp2 to rhodanese and was time-dependent. Re-activation did not occur in the reaction mixture lacking rhodanese.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

The isolation and characterization of specific 3-methylcholanthrene-binding proteins from rat liver cytosol

The major proteins to which 3-methylcholanthrene specifically binds have been purified over 480-fold with a 45% yield compared to a rat liver 100,000g supernate. The procedure involved a batch ion-exchange technique together with hydrophobic gel filtration and chromatofocusing chromatography. The multiple, specific 3-methylcholanthrene-binding proteins obtained from this protocol had apparent isoelectric points of pH 6.3, 6.0, 5.7, and 5.5 on elution from a chromatofocusing column. They all shared a common sedimentation coefficient as determined by sucrose gradient analysis of 4.4 S. Gel filtration on Sephadex G-75 gave a common Stokes radius of 27 A. An analysis of these chromatofocusing peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed those which eluted at pH 6.3 and 6.0 to contain two major protein bands of Mr 32,000 and 34,000, together with several contaminating proteins. In contrast, the peaks from chromatofocusing which eluted at pH 5.7 and 5.5 contained three major proteins of Mr 40,000, 25,000, and 14,000. The specific binding capability of these chromatofocusing peaks was found to be unstable to temperatures of -30 degrees C and below. Competition studies showed that these proteins were not steroid receptors, and that only polycyclic aromatic hydrocarbons which could induce cytochrome P-450c were able to displace 3-methylcholanthrene from the binding site. A marked preference was noted for polycyclic aromatic hydrocarbons with four to five benzene rings arranged in a nonlinear fashion, suggesting the stereochemical requirements of the protein binding site. The stability of the noncovalent interaction between the proteins and 3-methylcholanthrene was in the range of pH 7 to 9.     

Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991].     

Structural requirements for cooperativity in ileal bile acid-binding proteins

Ileal bile acid-binding proteins (I-BABP), belonging to the family of intracellular lipid-binding proteins, control bile acid trafficking in enterocytes and participate in regulating the homeostasis of these cholesterol-derived metabolites. I-BABP orthologues share the same structural fold and are able to host up to two ligands in their large internal cavities. However variations in the primary sequences determine differences in binding properties such as the degree of binding cooperativity. To investigate the molecular requirements for cooperativity we adopted a gain-of-function approach, exploring the possibility to turn the noncooperative chicken I-BABP (cI-BABP) into a cooperative mutant protein. To this aim we first solved the solution structure of cI-BABP in complex with two molecules of the physiological ligand glycochenodeoxycholate. A comparative structural analysis with closely related members of the same protein family provided the basis to design a double mutant (H99Q/A101S cI-BABP) capable of establishing a cooperative binding mechanism. Molecular dynamics simulation studies of the wild type and mutant complexes and essential dynamics analysis of the trajectories supported the role of the identified amino acid residues as hot spot mediators of communication between binding sites. The emerging picture is consistent with a binding mechanism that can be described as an extended conformational selection model.     

Comparison of cytosol retinol binding proteins from bovine retina, dog liver, and rat liver

Cytosol retinol binding proteins (CRBP's) have been purified from rat liver, dog liver, and bovine retina. All had identical molecular weights on sodium dodecyl sulfate electrophoresis. They had different RF values on non-sodium dodecyl sulfate gels at pH 8.9. The three CRBP exhibited similar absorption and fluorescence spectra. The absorbance of the ligand was perturbed after binding, the main band shifting bathochromically and exhibiting a lambda(max) at 350 nm compared with 328 nm for free retinol in hexane. Additionally, subsidiary peaks appeared at 335 and 367 nm. Rabbit antiserum against rat liver CRBP cross-reacted with CRBP's from dog liver and bovine retina. The Ouchterlony immunodiffusion technique indicated that these proteins have molecular structures with identical antigenic determinants. All three CRBP's had amino acid composition that were virtually identical, as judged by our own observations and those of other laboratories. The molecular structure of cytosol retinol binding proteins appears to be highly conserved, irrespective of species or tissue of origin.     

Primary structure and cellular distribution of two fatty acid-binding proteins in adult rat kidneys

Fatty acid-binding proteins (FABPs) were purified from the kidneys of female and male rats and characterized by primary structure and histological distribution in the kidney. Two FABPs (14 and 15.5 kDa) were found in male rat kidney cytosol whereas only 14-kDa FABP could be recognized in female rat kidneys throughout the purification steps. The amino acid sequence of the 14-kDa FABP was identical to that of rat heart FABP deduced from the cDNA sequence (Heuckeroth, R. O., Birkenmeier, E. H., Levin, M. S., and Gordon, J. I. (1987) J. Biol. Chem. 262, 9709-9717). Structural analysis of the male-specific 15.5-kDa FABP identified this second FABP as a proteolytically modified form of alpha 2u-globulin, an 18.7-kDa major urinary protein of adult male rats (Unterman, R. D., Lynch, K. R., Nakhasi, H. L., dolan, K. P., Hamilton, J. W., Cohn, D. V., and Feigelson, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3478-3482) which shares a common ancestry with a number of hydrophobic ligand-binding proteins such as serum retinol-binding proteins. Immunohistochemical investigation disclosed that heart-type FABP (14-kDa FABP) is localized in the cytoplasm of the epithelia of the distal tubules in both male and female rat kidneys whereas 15.5-kDa FABP immunostaining was observed predominantly in the endosomes or lysosomes of proximal tubules in male rat kidneys. These results suggest strongly the functional divergence of two FABPs in the rat kidney.