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Two radiolabeled hepatocarcinogens, N,N-dimethyl-4-aminoazobenzene (DAB) and 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB), were rapidly cleared from the blood of rats after i.v. administration, with half-lives of 40 and 70 sec, respectively. Rates of hepatic uptake and biliary secretion of [14C]-3'-Me-DAB were double that of [14C]DAB within 30 min of administration. Two hr after azo dye injection, the hepatic output into bile of [14C]-3'-Me-DAB-derived radioactivity was three times that of [14C]DAB. Fifty and 75% of the total 3'-Me-DAB-derived radioactivity was recovered in blood, liver, and bile 30 and 120 min after injection while only 30 to 40% of the administered [14C]DAB-derived radioactivity was recovered at these times. We postulate the existence of an extrahepatic azo dye accumulation site which may compete with the ability of the liver to clear azo dye from the circulation and which releases 3'-Me-DAB-derived radioactivity more readily than that of DAB. Azo dye metabolites were isolated from liver, bile, and blood. The chromatographic pattern of liver metabolites generated in vivo by rats which received either hepatocarcinogen was obtained and compared with that of biliary metabolites. With either azo dye, some metabolites were located exclusively in the liver, some were secreted immediately into bile, while others were present in both liver and bile, indicating selectivity in biliary excretion.

Eight-week-old rats had twofold higher hepatic ligandin concentration than 10-day-old animals as determined immunologically and by steroid isomerase and glutathione S-transferase assays. Increased ligandin content was accompanied by parallel increase in subunit synthesis as determined by [3H]leucine incorporation into each subunit relative to incorporation into total cytosolic proteins. The mRNA content for each ligandin subunit was twofold higher in older animals as determined by cell-free in vitro translation followed by immunoprecipitation and dot hybridization using a ligandin cDNA probe. When poly A mRNA from the postmitochondrial fraction of liver from young or old rats was subjected to agarose gel electrophoresis under denaturing conditions and hybridized to ligandin cDNA probe, a single 11 S band was obtained. With RNA from total liver, an additional 13 S band was obtained, suggesting the existence of a precursor form of ligandin mRNA. Since precursor polypeptides were not observed with RNA from total liver in cell-free in vitro translation systems, the precursor form requires processing to the 11 S form before the mRNA becomes functional.

Following administration of phenobarbital to rats, liver ligandin content, bilirubin binding, glutathione-S-transferase and steroid isomerase activities increased by 150% and the 22000-dalton subunit was selectively increased. Following administration of 3'-methyl-N,N-dimethyl-4-aminoazobenzene, rat liver ligandin content and steroid isomerase decreased by 65%, glutathione-S-transferase increased by 100%, bilirubin binding was abolished, and the relative proportion of the 22000- and 25000-dalton subunits remained unchanged.

Small amounts of metabolite-binding protein (MBP) originally characterized from the bile were detected in rat serum and cytosol by an indirect enzyme-linked immunoabsorbant assay. The site of MBP synthesis was shown to be the liver based upon results of (1) the in vitro translation of liver poly(A)+ mRNA, followed by immunoprecipitation with anti-MBP sera and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate, and (2) immunoprecipitation of bile collected from [3H]leucine perfused liver in situ and SDS-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate. To determine whether part of the MBP in bile is derived from the circulation, [125I]MBP was injected intravenously and bile was collected and subjected to SDS-polyacrylamide gel electrophoresis and fluorography. Intact [125]MBP was not detected in bile even though several other iodinated bile proteins were taken up by the liver from the circulation and secreted intact into the bile under similar experimental conditions. These data indicate that MBP is synthesized in the liver and secreted into the bile and circulation independently. In addition, MBP was not found in brain, spleen or kidneys.

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 lambda gt11. 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 pulse-chase technique was employed to determine the synthesis of the subunits of ligandin (glutathione S-transferase 1-2) by isolated hepatocytes. Ligandin comprised 2.5-3% of the total proteins synthesized. A slightly higher incorporation of [35S]methionine into the 22 k than the 25 k subunit was observed. However, the ratio of [35S]methionine incorporation into the subunits remained constant throughout the chase period, suggesting that, in spite of the considerable sequence homology, the conversion of 25 k to 22 k subunit does not occur in vivo.

When liver cytosol prepared from rats administered [14C]3'-methyl-N,N-dimethyl-4-aminoazobenzene was subjected to Sephadex gel chromatography, four peaks (I-IV) of radioactivity containing proteins and one peak (V) of radioactivity devoid of protein were obtained. Forty to fifty-five per cent of the radioactivity in the protein peaks was butanol-extractable. When the protein peaks were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, over 90% of the radioactivity was separated from the proteins, indicating lack of covalent binding. Several differences in the metabolite patterns were seen when the butanol-extractable metabolites from the five chromatographic peaks were analyzed by TLC. When pooled fractions of the peaks were incubated with isolated rat liver nuclei, only radioactivity associated with peak II was translocated into the nucleus. Translocation was time- and temperature-dependent and was maximal at 40 min at 37 degrees C. Only 10 to 12% of the radioactivity associated with peak II could be translocated even in the presence of an excess of nuclei, indicating that specific protein metabolite adduct(s) present in this fraction is/are translocated. Five per cent of translocated radioactivity was irreversibly bound to DNA, 3% to RNA, 67% to non-histone proteins, and 7.5% to histones; the remaining was not associated with any of these macromolecules.

Uptake, metabolism, and biliary secretion of 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) were studied in isolated rat liver which was perfused with protein-free fluorocarbon medium. [14C]3'-Me-DAB (5-10 nmol) was injected into the portal vein and allowed to recirculate. The recovery of radioactivity in bile was 7.5, 14, and 20% at 15, 30, and 45 min of injection, respectively. At 45 min, the liver contained an additional 17% of injected radioactivity. Azo dye metabolites in perfused liver differed from those in vivo; metabolites co-migrating with 3'-CHO-DAB and 3'-methyl-n,n-methyl aminoazobenzene (Me-MAB) (and the parent compound 3'-Me-DAB) were present, while metabolites co-migrating with 3'-Me-4'-OH-AB and 3'-CH2OH-MAB were increased and metabolites co-migrating with 3'-CH2OH-DAB were decreased. In bile from perfused liver, metabolites co-migrating with 3'-CHO-DAB and 3'-Me-MAB were undetectable. When proteins from normal rat bile were injected into portal vein 15 min after the administration of 3'-Me-DAB, the compounds co-migrating with 3'-Me-MAB, 3'-CHO-DAB, 3'-Me-4'-OH-AB, and 3'-CH2OH-MAB decreased, and compounds co-migrating with 3'-CH2OH-DAB increased in the liver; in bile, there was increase in 3'-Me-MAB, 3'-CHO-DAB, and 3'-Me-4'-OH-MAB, which there was a decrease in N-Ac-3'-Me-4'-OH-AB and 3'-COOH-DAB. Appearance of protein-metabolite adducts in bile was also observed after addition of normal bile proteins to the perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Bilirubin glucuronoside glucuronosyltransferase (EC 2.4.1.95) converts bilirubin monoglucuronide to bilirubin diglucuronide and is concentrated in plasma membrane-enriched fractions of rat liver homogenates. The enzyme was purified 2,000-fold to homogeneity from rat liver. The pI of the enzyme is 7.9 +/- 0.2. The enzyme has a molecular weight of 160,000 and is an oligomer of 28,000 dalton subunits. Km for purified enzyme was 35 microM and Vmax was 2.2 mumol of bilirubin diglucuronide formed/min/mg of protein. Freshly biosynthesized bilirubin monoglucuronide was injected intravenously into homozygous Gunn rats which had bile duct cannulation. Gunn rats lack UDP-glucuronate glucuronyltransferase activity (EC 2.4.1.17), have normal bilirubin glucuronoside glucuronosyltransferase activity, cannot form bilirubin monoglucuronide in vitro or in vivo, and do not excrete bilirubin glucuronides after intravenous injection of unconjugated bilirubin. Within 1 h, approximately 75% of the injected conjugated bilirubin was recovered in bile, of which 20% consisted of bilirubin diglucuronide. These results indicate that bilirubin glucuronide glucuronosyltransferase catalyzes conversion of bilirubin monoglucuronide to diglucuronide in vivo.

1. The lipoproteins of the liver cell sap can be resolved by paper electrophoresis into five components. Almost two-thirds of the total lipid, cholesterol and phospholipid are present in the slowest- and the fastest-moving components. 2. There is a two- to three-fold increase in the lipid content of the liver cell sap after the administration of carbon tetrachloride. 3. The cholesterol and phospholipid contents of the serum alpha- and beta-lipoproteins separated by paper electrophoresis fall after treatment with carbon tetrachloride, whereas the total lipid content of that fraction that remains at the origin rises.

Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier ( KM = 5.3 µ m), which is competitively inhibited by cyanidine 3-glucoside ( Ki = 51.6 µ m) and mainly noncompetitively by cyanidin ( Ki = 88.3 µ m). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site ( Kd = 1.7 n m) in the carnation carrier. The other antibody identified a high-affinity binding site for cyanidine 3-glucoside ( Kd = 1.7 µ m) on the carnation carrier only, and a high-affinity bilirubin-binding site ( Kd = 0.33 n m) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabelled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites. [ABSTRACT FROM AUTHOR]

In the 1960s, my lab was interested in understanding how bilirubin and other organic anions are transferred from the plasma through the liver cell and into the bile. We performed [...]