Characteristics of the cell surface antigen, p72, associated with a variety of human tumours and mitogen-stimulated T-lymphoblasts

We recently purified two closely related 33 kDa proteins from rat hepatic cytosol, designated bile acid binder I and II, which selectively bind bile acids with comparable affinity as glutathione S-transferase B. This work has now been extended to human liver in which we have identified a similar cytosolic binding activity in the 30-40 kDa fraction from gel filtration. Subsequent chromatofocusing and hydroxyapatite chromatography resulted in the isolation of a homogeneous monomeric protein of 36 kDa. The binding affinity of this protein for lithocholate using the displacement of 1-anilino-8-naphthalenesulfonate (ANS) was 0.1 microM, whereas human hepatic glutathione S-transferases purified from glutathione affinity chromatography demonstrated no competitive displacement of ANS.     

Comparison of the effects of bile acids and GSH on the fluorescence of bound 1-anilino-8-naphthalene sulfonate and the enzymatic activity of cationic and neutral human hepatic GSH S-transferases

Three cationic (C1, C2, A1) and a neutral (N1) glutathione (GSH) S-transferase were purified to homogeneity from human liver, as we have previously reported. GSH had no effect on the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS) bound by transferase C1 and N1, but markedly enhanced the fluorescence with C2 and A1 without changing the affinity for ANS. This effect of GSH was saturable and with C2 was intermediate between A1 and C1. Bile acids inhibited the fluorescence of ANS bound to C1 and C2. GSH in the presence of bile acids further decreased the fluorescence of ANS bound to C1 and increased the fluorescence with C2. Transferase A1 showed decreased fluorescence in the presence of lithocholic acid and increased fluorescence in the presence of cholic acid; both changes were reversed by GSH. Transferase N1 showed increased fluorescence of bound ANS in the presence of various bile acids and this effect was diminished in the presence of GSH. Enzyme activity of the transferase was inhibited by bile acids with the exception of transferase A1. All the proteins bound lithocholic acid. The inhibition of C1 and N1 was greater at pH 6.5 than 7.4 and the order of addition of substrates and inhibitor made no difference.     

Binding of bile acids, oleic acid, and organic anions by rat and human hepatic Z protein

Binding affinities of purified Z proteins from rat and human liver for bile acids, oleic acid, and organic anions were studied. Purification of Z protein from both rat and human hepatic cytosol was performed by gel filtration, chromatofocusing, and hydroxyapatite chromatography. Both purified proteins showed the same molecular weight (Mr = 14,000) and isoelectric points were 6.9 and 6.5 for rat and human proteins, respectively. Binding studies were performed by the competitive displacement of 1-anilino-8-naphthalene sulfonate. Rat and human Z proteins exhibited similar binding affinities for bile acids, oleic acid, and organic anions. Among various bile acids, both proteins bound monohydroxy bile acids with high affinity and trihydroxy bile acids with low affinity; sulfates were bound with higher and glucuronides with lower affinity than their parent bile acids. In comparison with GSH S-transferases, rat Z protein had lower affinity for bile acids than rat GSH S-transferase B and human Z protein had higher affinity for bile acids than human cationic GSH S-transferase. The role for Z protein in the intracellular binding of bile acids may be particularly important in human liver.     

Comparison of the affinities of newly identified human bile acid binder and cationic glutathione S-transferase for bile acids

The bile acid binding properties of the newly identified bile acid binder (Mr = 36,000) (FEBS Lett. 1984. 177: 31-35) and the major cationic glutathione (GSH) S-transferase (Mr = 50,000) in human liver cytosol were compared. Binding affinities were measured by the competitive displacement by bile acids of 1-anilino-8-naphthalene sulfonate (ANS) bound to the proteins and, in some cases, by direct methods of flow dialysis and equilibrium dialysis. The binding affinities for various bile acids by the human bile acid binder were 2-5 orders of magnitude greater than those by human cationic GSH S-transferase. This suggests an important physiologic role for the former protein in intracellular transfer of bile acids in human liver.     

Comparison of the binding sites of GSH S-transferases of the Ya- and Yb-subunit classes: effect of glutathione on the binding of bile acids

We have previously observed that the Ya subunit-containing glutathione (GSH) S-transferases from rat liver exhibit a common high affinity binding site for lithocholic acid, bilirubin, and sulfobromophthalein (BSP) (1984. J. Lipid Res. 25: 1177-1183). Subsequently we found that cholic acid and its amidates bound to a site on the Ya subunit separate for the lithocholic acid/bilirubin site (1986. J. Lipid Res. 27: 955-966). We now have extended this work by showing that amidates of lithocholic acid as well as chenodeoxycholic acid and its amidates competitively displace [14C]lithocholic acid from the Ya subunit. GSH did not inhibit binding of any of the ligands to the high affinity Ya site, but did inhibit binding to the cholic acid site on the Ya subunit. We have also defined the binding sites and effects of GSH on the Yb class of subunits. Lithocholic, chenodeoxycholic, and cholic acids (and amidates) shared a common site on the Yb or Y'b subunit, whereas BSP and bilirubin were bound at a different site. Both the bile acid and organic anion sites on the Yb subunit were inhibited by GSH. The inhibition by GSH in all cases (Ya cholic acid site or Yb bile acid or bilirubin sites) was saturable, of the competitive type, and incomplete at maximal GSH concentrations, suggesting that when GSH binds to its distinct substrate site, it induces a conformational change in the proteins affecting the other binding sites.     

Binding of bile acids by glutathione S-transferases from rat liver

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.     

Identification of two lithocholic acid-binding proteins. Separation of ligandin from glutathione S-transferase B.

1. Two lithocholic acid-binding proteins in rat liver cytosol, previously shown to have glutathione S-transferase activity, were resolved by CM-Sephadex chromatography. 2. Phenobarbitone administration resulted in induction of both binding proteins. 3. The two proteins had distinct subunit compositions indicating that they are dimers with mol.wts. 44 000 and 47 000. 4. The two lithocholic acid-binding proteins were identified by comparing their elution volumes from CM-Sephadex with those of purified ligandin and glutathione S-transferase B prepared by published procedures. Ligandin and glutathione S-transferase B were eluted separately, as single peaks of enzyme activity, at volumes equivalent to the two lithocholic acid-binding proteins. 5. Peptide 'mapping' revealed structural differences between the two proteins.     

Binding of bile acids by 100 000g supernatants from rat liver.

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.     

A novel binding site for bile acids on human serum albumin

Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthelene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum album whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (K_d) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 μM, respectively, which was measured by equilibrium dialysis at 37° C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.     

Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin

Binding of lithocholic acid, bilirubin, and gossypol to glutathione S-transferase B (ligandin or transferase YaYc) was compared using four methods. Tryptophan quenching revealed a single high affinity site for bilirubin and gossypol but could not be used for lithocholic acid. Both displacement of the fluorescent probe, 1-anilino-8-naphthalenesulfonate, and spectral changes induced by bilirubin binding demonstrated a common high affinity site for which all three ligands compete. Similar results were obtained by equilibrium dialysis. The dissociation constants for the binding of both bilirubin and lithocholic acid were comparable with the various methods (range 0.2-0.7 microM). Thus, lithocholic acid and bilirubin share a high affinity binding site on gluthathione S-transferase B that appears to be separate from the binding site for substrates.     

Cholic acid binding by glutathione S-transferases from rat liver cytosol.

Cholic acid-binding activity in cytosol from rat livers appears to be mainly associated with enzymes having glutathione S-transferase activity; at least four of the enzymes in this group can bind the bile acid. Examination of the subunit compositions of different glutathione S-transferases indicated that cholic acid binding and the ability to conjugate reduced glutathione with 1,2-dichloro-4-nitrobenzene may be ascribed to different subunits.     

A comparison of the glutathione S-transferases of trout and rat liver.

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.     

Rat lung glutathione S-transferases: subunit structure and the interrelationship with the liver enzymes

Six forms of glutathione S-transferases designated as GSH S-transferase I (pI 8.8), II (pI 7.2), III (pI 6.8), IV (pI 6.0), V (pI 5.3) and VI (pI 4.8) have been purified from rat lung. GSH S-transferase I (pI 8.8) is a homodimer of Mr 25,000 subunits; GSH S-transferases II (pI 7.2) and VI (pI 4.8) are homodimers of Mr 22,000 subunits; and GSH S-transferases III (pI 6.8), IV (pI 6.0) and V (pI 5.3) are dimers composed of Mr 23,500 and 22,000 subunits. Immunological properties, peptide fragmentation analysis, and substrate specificity data indicate that Mr 22,000, 23,500 and 25,000, are distinct from each other and correspond to Ya, Yb, and Yc subunits, respectively, of rat liver.     

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively.     

Hepatic glutathione S-transferases in rainbow trout and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

Glutathione S-transferase in the cytosol of rainbow trout liver was partially purified by affinity chromatography on a column with glutathione coupled to epoxy-activated Sepharose 6B, which retained 94% of the total activity. Chromatofocussing on a Polybuffer exchanger 118 column separated the glutathione S-transferase into six major cationic isoenzymes (K1-K6), and some minor fractions. SDS-polyacrylamide slab gel electrophoresis showed K1-K3 to be heterodimers with subunits of Mr 25,000 and 26,500, and K4-K6 to be homodimers with subunits of Mr 25,000. The glutathione S-transferase isoenzymes were partially characterized by different biochemical parameters. The hepatic rainbow trout glutathione S-transferases were inhibited by the organic water pollutants, 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid. The same kinetic inhibition patterns were observed with these inhibitors as for rat liver glutathione S-transferases. It is concluded that rainbow trout glutathione S-transferases can play a key role in the detoxication of organic micropollutants in the aquatic environment.     

Lithocholate binding by Y and Y' proteins in bovine small intestine.

Cytosolic proteins may play an important role in the intracellular transport of bile acids in enterocytes. The lithocholate binding properties of cytosolic protein from bovine small intestine were studied. Lithocholate binding was observed in the Y (45-50 kDa), Y' (30-35 kDa), and Z fractions (10-15 kDa) following gel filtration of cytosol. A Y protein with glutathione S-transferase activity (46 kDa) was purified by S-octyl-glutathione affinity chromatography and chromatofocusing (eluted at pH 7.5) of the Y fraction. Two Y' bile acid binding proteins with dihydrodiol dehydrogenase activity were partially purified from the Y' fraction by chromatofocusing and hydroxyapatite-HPLC. The lithocholate binding affinity of Y' protein (Kd < 0.35 microM) was higher than that of Y protein (Kd = 2 microM) and was comparable to that of Z protein (Kd = 0.2 microM). The binding affinity of Y protein was higher for bilirubin (Kd = 2.5 microM) than that for BSP (Kd = 200 microM). This was comparable to the binding affinity of bovine hepatic Y protein. These data indicate that Y' and Z proteins participate in the intracellular transport of bile acids from the brush border to the basolateral pole in enterocytes.     

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated (N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg²⁺ and its optimum pH was about pH 7.0. In term of maximum velocity (V_max) and the Michaelis constant (K_m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.     

Purification and characterization of a new form of glutathione S-transferase from human erythrocytes

Presence of a new form of glutathione S-transferase has been demonstrated in human erythrocytes. using two different affinity ligands this enzyme has been separated from the previously characterized glutathione S-transferases ?. The new enzyme is highly basic with a pI of > 10. The new enzyme which represents less than 5 percent of glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene as substrate and about 10 percent of total glutathione S-transferase protein of erythrocytes has different amino acid composition, substrate specificities, and immunological characteristics from those of the major erythrocyte glutathione S-transferase ?. Immunological properties of the new enzyme indicate that this form may be different from other glutathione S-transferases of human tissues.     

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.     

Ligand specificity and conformational stability of human fatty acid-binding proteins.

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.