Liver Ligandin Research Articles

Isolation of a human liver ligandin cDNA clone and demonstration of seouence homology at ligandin loci in rats and humans

Using a monospecific antibody to the major cytosolic glutathione-S-transferase of human liver, we have isolated a cDNA clone from a human liver cDNA expression vector library in λ gtll. The clone cross-hybridizes with a rat liver ligandin (glutathione-S-transferase 1–2) cDNA probe. The clone has an insert of 1.25 kb, a size sufficient to code for the 23 kilodalton subunit of human GST. Digestion of the insert with Hinf I produced three fragments (0.8 kb, 0.4 kb and 0.1 kb). A similar pattern of multiple bands was observed when rat liver GST1-2 cDNA probe was used for Southern blot analysis of Pst digests of rat and human genomic DNAs. These data suggest that these two functionally similar proteins exhibit sequence homology between their respective cDNAs and at ligandin loci, in spite of the lack of immuno-crossreactivity between them.

The dependence of biliary methylmercury secretion on liver GSH and ligandin

The biliary secretion of methylmercury was investigated in male rats which were given i.p. 400 μmoles/kg azathioprine or 96 μmoles/kg benziodarone 2 hr after the i.v. injection of 5 μmoles/kg MeHgCl. A group of rats were given 400 mg/kg trans-stilbene oxide (TSO) for 4 days before treatment with 10 μmoles/kg MeHgCl. A common link between these three compounds is their interference with ligandin. Azathioprine is a competitive inhibitor of glutathione S-transferase, benziodarone is covalently bound to ligandin and TSO is an inducer of liver ligandin. Although only azathioprine depletes liver GSH stores, both azathioprine and benziodarone inhibited the biliary secretion of methylmercury. As there is published proof that the reaction of MeHg + with GSH does not require enzymatic help, the inhibitory effect of azathioprine and benziodarone confirms the role of ligandin in the transport of methylmercury or its GSH complex. However, the biliary secretion of methylmercury was increased ortly slightly by TSO pretreatment, but when 2hr after the injection of MeHgCl animals received 2 mmoles/kg GSH, secretion increased twice as much in TSO pretreated than in control rats. This indicates the dual dependance of biliary methylmercury secretion on liver GSH and ligandin.

Glutathione S-transferase in human hepatocellular carcinoma.

Qualitative and quantitative changes in glutathione S-transferase (GSH-T) were studied in human hepatocellular carcinoma. GSH-T specific activity (mumoles per min per mg protein) was variably reduced in hepatocellular carcinoma. Similar changes were seen in "cationic" GSH-T (ligandin) concentration determined by radioimmunoassay. Immunohistochemical studies with antihuman liver ligandin suggest that positive staining was more frequently found in well-differentiated tumors. The relative activities of "cationic," "neutral," and "anionic" transferases (pI greater than 7.5) activity ranged from virtually absent to near normal values. "Neutral" (pI 6 to 6.5) and "anionic" (pI less than 5.4) species were present more often in tumors than in normal liver. In two cases, normal liver tissue and tumor were obtained from the same patient. In one, only quantitative differences were present, while in the other "cationic" and "neutral" GSH-Ts were present in the normal liver tissue while both "cationic" and "anionic" species were found in the tumor. Our studies indicate that qualitative as well as quantitative changes of GSH-T occur in human hepatocellular carcinoma.

The effect of phenobarbital and 3′-methyl- N, N-dimethyl-4-aminoazobenzene administration on catalytic, binding and immunological properties of ligandin subunits in rat liver

Following administration of phenobarbital to rats, liver ligandin content, bilirubin binding, glutathione- S-transferase and steriod isomerase activities by 150% and the 22 000-dalton subunit was selectively increased. Following adminstration of 3′-methyl- N, N-dimethyl-4-aminoazobenzene, rat liver ligandin content and steroid isomerase decrased by 65%, glutathione- S-transferase incrased by 100%, bilirubin binding was abolished, and the relative proportion of the 22 000- and 25 000-dalton subunits remained unchanged.

Bilirubin binding to human liver ligandin (glutathione S-transferase).

The number of binding sites and the dissociation constants were determined for the binding of bilirubin to human liver ligandin and to human serum albumin. Albumin has a primary bilirubin binding site (KD = 0.03 microM), measured by the peroxidase procedure, and two apparently equivalent secondary binding sites (KD = 2 microM), determined by fluorescence quenching experiments. By contrast, ligandin does not have a corresponding high affinity site. The absence of this high affinity site was shown both by the peroxidase procedure and by direct competition between albumin and ligandin for bilirubin. Bilirubin binding to ligandin, measured by fluorescence quenching, is complex. At both pH 6.5 and 7.4, two interacting sites were observed with a Hill coefficient of 1.5, K' approximately 5 microM. Bilirubin binding to ligandin is not independent of glutathione S-transferase activity. Depending upon pH and upon the order with which the reactants are added, bilirubin can markedly alter the transferase activity. The results are interpreted in terms of kinetically stable conformational isomers of ligandin induced by bilirubin or by glutathione.

Subunit composition, organic anion binding, catalytic and immunological properties of ligandin from rat testis.

Rat testicular and liver cystols contain ligandin as determined immunologically, and have high glutathione-S-transferase activity. Unlike liver cytosol, testicular cytosol does not contain protein components that bind bilirubin or sulfobromophthalein with high affinity. To investigate these effects, ligandin was purified to homogeneity from rat testis. Whereas rat liver ligandin consists of equal amounts of two subunits with molecular weights of 22,000 (Ya) and 25,000 (Yb), more than 90% of testicular ligandin consists of Yb. Rat testicular ligandin is immunologically similar to liver ligandin, and has identical glutathione-S-transferase activity, but lacks the capacity for high affinity binding of bilirubin and sulfobromophthalein. The amino acid composition and other properties of testicular ligandin are similar to those of the Yb subunit of liver ligandin. Sulfobromophthalein and bilirubin biphasically inhibit the glutathione-S-transferase activity of liver ligandin: initial high affinity inhibition is followed by reduced inhibition. Testicular ligandin has only low affinity inhibition kinetics. These results suggest that Ya is required for high affinity binding, and that reduced organic anion binding by testicular ligandin results from the lower amounts of Ya in testis.

Structural, catalytic, binding, and immunological properties associated with each of the two subunits of rat liver ligandin.

Structural, catalytic, binding, and immunological properties associated with each of the two subunits of rat liver ligandin.

Glucocorticoid binding in the chicken liver cytosol. Characterization of five macromolecular binding components.

The heterogeneity of the glucocorticoid binding in the chicken liver cytosol, previously suggested by the results obtained with crude preparations, was confirmed using different techniques such as stepwise ammonium sulfate precipitation, hydroxyapatite chromatography and gel filtration on Sephadex G-200. The latter method permitted the separation of five glucocorticoid binding macromolecules respectively named binders S1, S2, S3, S4 and S5, according to their decreasing apparent molecular size upon gel filtration. Apparent molecular weight, binding affinity, capacity and specificity of these five moieties were examined. In addition, S-aryl-transferase activity using glutathione as co-substrate was studied and found to coincide mostly with the fractions containing binder S4, which might represent an avian liver ligandin.

Separate identities of ligandin and the [formula omitted]-protein, a major protein to which carcinogenic hydrocarbons are covalently bound

There is a protein moiety in the C3H mouse liver cytosol which gives a line of identity with rat liver ligandin one of three azo dye binding proteins of the liver using anti-rat ligandin. This mouse liver protein has been termed mouse ligandin and is not the h -protein, the major target protein in the mouse liver of methylcholanthrene and its metabolites. Mouse ligandin is identical to a minor methylcholanthrene binding protein species that was found previously to consist of basic proteins II and III. Both mouse ligandin and mouse h -protein contain glutathione S-transferase activity with different substrate specificitles.

Interactions of azodye carcinogens and azodye carcinogen conjugates with specific proteins in the rat liver

There is a protein moiety in the C3H mouse liver cytosol which gives a line of identity with rat liver ligandin one of three azo dye binding proteins of the liver using anti-rat ligandin. This mouse liver protein has been termed mouse ligandin and is not the h-protein, the major target protein in the mouse liver of methylcholanthrene and its metabolites. Mouse ligandin is identical to a minor methylcholanthrene binding protein species that was found previously to consist of basic proteins II and III. Both mouse ligandin and mouse h-protein contain glutathione S-transferase activity with different substrate specificitles.