Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes.

Physiological properties of isolated pairs of rat hepatocytes were examined within 5 h after dissociation. These cells become round when separated, but cell pairs still display membrane specializations. Most notably, canaliculi are often present at appositional membranes which are flanked by abundant gap and tight junctions. These cell pairs are strongly dye-coupled; Lucifer Yellow CH injected into one cell rapidly diffuses to the other. Pairs of hepatocytes are closely coupled electrically. Conductance of the junctional membrane is not voltage sensitive: voltage clamp studies demonstrate that gj is constant in response to long (5 s) transjunctional voltage steps of either polarity (to greater than +/- 40 mV from rest). Junctional conductance (gj) between hepatocyte pairs is reduced by exposure to octanol (0.1 mM) and by intracellular acidification. Normal intracellular pH (pHi), measured with a liquid ion exchange microelectrode, was generally 7.1-7.4, and superfusion with saline equilibrated with 100% CO2 reduced pHi to 6.0-6.5. In the pHi range 7.5-6.6, gj was constant. Below pH 6.6, gj steeply decreased and at 6.1 coupling was undetectable. pHi recovered when cells were rinsed with normal saline; in most cases gj recovered in parallel so that gj values were similar for pHs obtained during acidification or recovery. The low apparent pK and very steep pHi-gj relation of the liver gap junction contrast with higher pKs and more gradually rising curves in other tissues. If H+ ions act directly on the junctional molecules, the channels that are presumably homologous in different tissues must differ with respect to reactive sites or their environment.

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata.

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Australia antigen: detection and transmission in shellfish.

Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

Alteration of bile canalicular enzymes in cholestasis. A possible cause of bile secretory failure.

Bile secretory failure (cholestasis) may result from several possible mechanisms involved in bile secretion. We have examined the possibility that abnormalities in enzyme content, composition, and turnover of liver plasma membrane constituents are altered in cholestasis. Severe and mild cholestasis were produced by 5 days of bile duct ligation and ethinyl estradiol administration, respectively. Bile duct ligation but not ethinyl estradiol treatments was associated with elevations of the serum bilirubin level and 5'-nucleotidase activity. However, basal bile flow and bilirubin transport maximum (T(m)) were significantly reduced after ethinyl estradiol treatment. Liver plasma membrane fractions rich in canalicular membranes were prepared from groups of rats in each of three categories; normal, after bile duct ligation, or ethinyl estradiol administration, and their respective controls. Electron microscopy and enzyme marker studies demonstrated plasma membrane fractions free of significant contamination. Plasma membrane fractions prepared from mild as well as severe cholestasis had increased alkaline phosphatase activity, and reduced 5'-nucleotidase and Mg(2+)-ATPase activities. Co(2+)-CMPase activity was unchanged. Kinetic analysis of 5'-nucleotidase and Mg(2+)-ATPase activities in plasma membrane fractions demonstrated reduced V(maz) (but unaltered K(m)). Reducted V(maz) was unrelated to addition in vitro of di-or trihydroxy bile salts or ethinyl estradiol and, therefore, suggests that reduced activities in cholestasis are due to decreased enzyme content. Cholestasis was not associated with changes in the synthesis or degradation rate of pulse-labeled plasma membrane proteins or alterations in the major protein bands separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Plasma membrane cholesterol, phospholipid, and neutral sugar content was unaltered, but sialic acid content was significantly increased in both forms of cholestasis. Alterations in specific canalicular enzymes in two forms of cholestasis suggest that these changes may be involved in the pathogenesis of bile secretory failure, or may result from cholestasis.

Immunological studies of Y protein. A major cytoplasmic organic anion-binding protein in rat liver.

An antibody produced against rat Y protein, the major cytoplasmic organic anion-binding protein in liver, was characterized. The antibody precipitated Y protein from liver supernatant fractions and specifically removed the organic anion-binding capacity from this fraction.Y protein was detected by immunodiffusion with this antibody in the supernates of rat liver, kidney, and small intestinal mucosa and was not detected in supernates of 16 other tissues including bile and serum. Precipitation with anti-Y was not detected with supernates of liver from 20 other species, including man. Quantitative radial immunodiffusion revealed Y protein to constitute 4.5% of supernatant protein in rat liver and approximately 2% of supernatant protein in rat kidney and small intestinal mucosa. Phenobarbital administration increased the concentration of Y protein in rat liver by 280%, but not in kidney or small intestinal mucosa, and was associated with increased plasma disappearance of sulfobromphthalein sodium, indocyanine green, and bilirubin, and increased hepatic, but not kidney or small intestinal mucosal, content of these organic anions. These observations provide further evidence indicating that the concentration of Y protein is a major determinant of organic anion flux across the plasma membrane of the liver cell.Immunodiffusion and immunoelectrophoresis revealed serological identity between Y protein, cortisol metabolite-binding protein I. and the major azocarcinogen-binding protein.

Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions.

Two hepatic cytoplasmic protein fractions, designated Y and Z, which bind sulfobromophthalein (BSP), bilirubin, and other organic anions, have been separated by G75 Sephadex gel filtration. The physiologic role of these protein fractions has been investigated. They are present in the 110,000 g supernatant fraction from the livers of all the species tested (rats, mice, guinea pigs, Rhesus monkeys, sheep, and man). Tissues which do not preferentially extract BSP or bilirubin from plasma do not contain these fractions, with the exception of small intestinal mucosa which contains Z. Anion binding by Y and Z fractions is not due to contamination with albumin. These fractions are responsible for the cytoplasmic localization of bilirubin in Gunn rats, and the fractions bind bilirubin, BSP, or indocyanine green (ICG), whether given in vivo or added in vitro to liver supernate from normal rats. Flavaspidic acid-N-methylglucaminate, bunamiodyl, and iodipamide, drugs known to interfere with the hepatic uptake mechanism, compete with bilirubin and BSP for binding to Z. These proteins appear to be important in the transfer of organic anions from plasma into the liver and provide a tool for the investigation of hepatic uptake mechanisms.

Structural and functional studies of ligandin, a major renal organic anion-binding protein.

Sephadex gel filtration of the 1000,000 g supernate of homogenates of rat kidney revealed binding of various organic anions (penicillin, Bromsulphalein [BSP], bilirubin, phenolsulfonphthalein [PSP], phlorizin, glutathione [GSH], p-amino hippurate (PAH), probenecid, conjugated bilirubin, and BSP-GSH) to a nonalbumin-containing protein fraction (Y), which precipated on addition of monospecific anti-rat liver ligandin (Y protein)-IgG, but not control IgG. Quantitatively similar organic anion binding was observed in vivo after injection of BSP, BSP-GSH, phlorizin, probenecid, conjugated bilirubin, PAH, or penicillin. The binding protein was purified to apparent homogeneity and is a basic protein (pI 8.9) of 44,000 daltons with two apparently identical subunits of 22,000 daltons. Monospecific antibody was produced against the renal protein. The results of binding studies in vivo and in vitro and phsicochemical, immunologic, structural, and binding site investigations indicate that the renal protein is identical to hepatic ligandin. Immunofluorescent studies utilizing anti-ligandin IgG previously localized ligandin in the kidney to all proximal tubular cells. By quantitative radial immunodiffusion, the concentration of renal ligandin was 31.2 plus or minus 2.2 mug/mg supernatant protein and was increased 160% above basal values by pretreatment of rats with tetrachloro-dibenzo-p-dioxin. Pretreatment with phenobarbital, DDT, or pregnene-16alpha-carbonitrile did not increase renal ligandin concentration but doubled hepatic ligandin concentration. Circular dichroism studies of renal ligandin revealed percent helical structure similar to hepatic ligandin and primary association contrasts were derived for BSP (10-6 M-1) and PAH, probenecid, and penicillin (10-3 M-1). Administration of BSP or probenecid simultaneously with [C14] penicillin resulted in increased plasma retention and reduced kidney and urinary bladder content of [14C] penicillin and a correlation coefficient of -0.8 between total kidney/plasma radioactivity and percent of protein-bound radioactivity bound to ligandin in the kidney. These studies indicate that renal and hepatic ligandin are identical. Their response to drugs and chemicals varies. Competitive binding between several organic anions for ligandin correlated with their renal uptake from plasma, which suggests that ligandin may function in the proximal tubular cell as a component of the renal organic anion transport system.

Taurocholate transport by rat liver canalicular membrane vesicles. Evidence for the presence of an Na+-independent transport system.

To elucidate the mechanism of vectorial translocation of bile acids in the liver, taurocholate transport was studied in isolated liver canalicular membrane vesicles by a rapid filtration method. The membrane vesicles revealed temperature-dependent, Na+-independent transport of taurocholate into an osmotically reactive intravesicular space. In the absence of sodium, taurocholate uptake followed saturation kinetics (apparent Km for taurocholate = 43 microM and Vmax = 0.22 nmol/mg protein X 20 s at 37 degrees C) and was inhibited by cholate and probenecid. Transstimulation by unlabeled taurocholate was also demonstrated. When the electrical potential difference across the membranes was altered by anion replacement, a more positive intravesicular potential stimulated, and a more negative potential inhibited, transport of taurocholate by the vesicles. Valinomycin-induced K+-diffusion potential (vesicle inside-positive) enhanced the rate of taurocholate uptake that was not altered by imposed pH gradients. These results indicate that rat liver canalicular plasma membrane contains a sodium-independent taurocholate transport system that translocates the bile acid as an anion across the membrane. In intact hepatocytes, the electrical potential difference across the canalicular membrane probably provides the driving force for taurocholate secretion. The contribution of nonionic diffusion to taurocholate secretion appears to be minimal.

Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia.

Bilirubin is conjugated with glucuronic acid in hepatocytes and subsequently secreted in bile. The major conjugate is bilirubin diglucuronide. Using sealed vesicles which are primarily derived from the canalicular (CMV) and sinusoidal (SMV) membrane vesicle domains of the plasma membrane of hepatocytes, we demonstrated that bilirubin glucuronides are transported by CMV by both ATP- and membrane potential-dependent transport systems. In CMV from normal rats, these processes are additive. In CMV from TR- rats, which have an autosomal recessively inherited defect in biliary secretion of nonbile acid organic anions, ATP-dependent transport of bilirubin diglucuronide was absent whereas the membrane potential driven system was retained. Other canalicular ATP-dependent transport systems, which were previously described for organic cations and bile acids, are functionally retained in TR- rats. Our study indicates that bilirubin glucuronides are primarily secreted into the bile canaliculus by an ATP-dependent mechanism which is defective in an animal model of the human Dubin-Johnson syndrome.

Studies of Y and Z, two hepatic cytoplasmic organic anion-binding proteins: effect of drugs, chemicals, hormones, and cholestasis.

The process by which various anions, including bilirubin and several dyes, drugs, hormones and their metabolites, are transferred from plasma into the liver cell is poorly understood. Two hepatic cytoplasmic proteins, Y and Z, that bind various organic anions in vivo and in vitro have been postulated to be involved in this process. The concentration of Y, the major organic anion-binding protein, increases in rat liver after administration of phenobarbital in association with enhanced organic anion transfer from plasma into liver as determined by initial plasma disappearance rate (K(1)) and hepatic dye content for sulfobromophthalein (BSP) and indocyanine green (ICG), as well as increased relative hepatic storage of BSP. Acute bile duct ligation failed to alter plasma disappearance or hepatic content of BSP in normal or phenobarbital-treated rats. Other drugs and chemicals which cause proliferation of hepatic smooth endoplasmic reticulum and enhancement of drug metabolism, such as allylisopropylacetamide, dieldrin, DDT, 3-methylcholanthrene, and benzpyrene increased Y and BSP K(1) and, where studied, hepatic BSP content. Alcohol feeding had no effect on Y, Z, or K(1) for BSP. Hypophysectomy and thyroidectomy increased Y but decreased K(1) and, where studied, hepatic content of BSP. Of several hormones studied, only thyroxine restored Y and K(1) to normal in hypophysectomized or thyroidectomized rats. Mice with congenital pituitary insufficiency also manifested increased Y which returned to normal after thyroxine administration. In hormone-deficient rats and mice, phenobarbital administration produced a further increase in Y suggesting that different mechanisms may be responsible for the change in Y resulting from drug administration and hormonal deprivation. Thyroxine, testosterone, or hydrocortisone did not alter BSP K(1) or Y in normal rats.Cholestasis produced by ethinyl estradiol administration or biliary obstruction reduced Y, Z, BSP K(1) and hepatic BSP content. These results support the hypothesis that Y and Z are involved in the transfer of BSP, ICG, and possibly other organic anions from plasma into the liver. The concentration of Y increased after administration of various drugs and chemicals as well as in thyroid deficiency. Thyroid hormone appears to be important in regulation of the intracellular concentration of Y. Because thyroid deficiency increased Y but decreased BSP K(1) and hepatic BSP content, other factors beside Y and Z influence hepatic organic anion uptake.

Isolation of multiple normal and functionally defective forms of uridine diphosphate-glucuronosyltransferase from inbred Gunn rats.

Gunn rats are a mutant strain of Wistar rats that have unconjugated hyperbilirubinemia due to absence of hepatic uridine diphosphate-glucuronosyltransferase (UDPGT; EC. 2.4.1.17) activity toward bilirubin. We isolated five UDPGT isoforms from solubilized microsomal fractions from liver of inbred Wistar (RHA) rats and congeneic Gunn rats. UDPGT isoform V (elution pH 7.5) from Wistar (RHA) rats is active toward bilirubin and 4'-hydroxydimethylaminoazobenzene. The corresponding isoform from Gunn rat liver was enzymically inactive but exhibited normal elution pH and mobility on NaDodSO4/polyacrylamide gel electrophoresis (Mr 53,000), and was recognized by a UDPGT-specific antiserum. UDPGT isoform I (elution pH 8.7) from Wistar (RHA) and Gunn rats was active toward 4-nitrophenol. The isoform from Gunn rat liver had only 10% of normal UDPGT activity, however UDPGT activity increased to normal upon addition of 15 mM diethylnitrosamine in vitro. Isoforms II (elution pH 8.4), III (elution pH 8.0), and IV (elution pH 7.8) from Gunn rats had normal UDPGT activities, except that Isoform IV was inactive toward bilirubin.

Hepatic conversion of bilirubin monoglucuronide to diglucuronide in uridine diphosphate-glucuronyl transferase-deficient man and rat by bilirubin glucuronoside glucuronosyltransferase.

The microsomal enzyme uridine diphosphate (UDP) glucuronate glucuronyltransferase (E.C. 2.4.1.17) catalyzes formation of bilirubin mono-glucuronide from bilirubin and UDPglucuronic acid. Bilirubin glucuronoside glucuronosyltransferase (E.C. 2.4.1.95), an enzyme concentrated in plasma membrane-enriched fractions of rat liver, converts bilirubin monoglucuronide to bilirubin diglucuronide. Bilirubin glucuronoside glucuronosyltransferase activity was studied in homogenates of liver biopsy specimens obtained from patients with the Crigler-Najjar syndrome (Type I) and in subcellular liver fractions of rats homozygous for UDP glucuronate glucuronyltransferase deficiency (Gunn strain). In patients with the Crigler-Najjar syndrome (Type I) and in Gunn rats, hepatic UDPglucuronate glucuronyltransferase activity was not measurable; however, bilirubin glucuronoside glucuronosyltransferase activity was similar to that in normal controls. The subcellular distribution of bilirubin glucuronoside glucuronosyltransferase activity in Gunn rat liver was similar to the distribution observed in normal Wistar rat liver.When bilirubin monoglucuronide was infused intravenously into Gunn rats, 29+/-5% of the conjugated bilirubin excreted in bile was bilirubin diglucuronide. After transplantation of normal Wistar rat kidney, which contained UDPglucuronate glucuronyltransferase activity, in Gunn rats, the serum bilirubin concentration decreased by 80% in 4 days. The major route of bilirubin removal was biliary excretion of conjugated bilirubin, approximately 70% of which was bilirubin diglucuronide. Although patients with the Crigler-Najjar syndrome (Type I) and Gunn rats lack UDP glucuronate glucuronyltransferase, their livers enzymatically convert bilirubin monoglucuronide to diglucuronide in vitro. Conversion in bilirubin monoglucuronide to diglucuronide was demonstrated in Gunn rats in vivo.

Bilirubin diglucuronide formation in intact rats and in isolated Gunn rat liver.

Bilirubin diglucuronide (BDG) may be formed in vitro by microsomal UDP glucuronosyl transferase (EC 2.4.1.17)-mediated transfer of a second mole of glucuronic acid from UDP-glucuronic acid, or by dismutation of bilirubin monoglucuronide (BMG) to BDG and unconjugated bilirubin, catalyzed by an enzyme (EC 2.4.1.95) that is concentrated in plasma membrane-enriched fractions of rat liver. To evaluate the role of these two enzymatic mechanisms in vivo, [(3)H]bilirubin mono-[(14)C]glucuronide was biosynthesized, purified by thin-layer chromatography, and tracer doses were infused intravenously in homozygous Gunn (UDP glucuronyl transferase-deficient) rats or Wistar rats. Bilirubin conjugates in bile were separated by high-pressure liquid chromatography and (3)H and (14)C were quantitated. In Gunn rats, the (14)C:(3)H ratio in BDG excreted in bile was twice the ratio in injected BMG. In Wistar rats the (14)C:(3)H ratio in biliary BDG was 1.25 +/- 0.06 (mean +/- SEM) times the ratio in injected BMG. When double labeled BMG was injected in Wistar rats after injection of excess unlabeled unconjugated bilirubin (1.7 mumol), the (14)C:(3)H ratio in BDG excreted in bile was identical to the ratio in injected BMG. Analysis of isomeric composition of bilirubin conjugates after alkaline hydrolysis or alkaline methanolysis indicated that the bile pigments retained the IX(alpha) configuration during these experiments. The results indicate that both enzymatic dismutation and UDP glucuronyl transferase function in vivo in BDG formation, and that dismutation is inhibited by a high intrahepatic concentration of unconjugated bilirubin. This hypothesis was supported by infusion of [(3)H]bilirubin-monoglucuronide in isolated perfused homozygous Gunn rat liver after depletion of intrahepatic bilirubin by perfusion with bovine serum albumin (2.5%), and after bilirubin repletion following perfusion with 0.34 mM bilirubin. From 20 to 25% of injected radioactivity was recovered in BDG in bile in the bilirubin-depleted state; only 8-10% of radioactivity was in BDG in bile after bilirubin repletion. After infusion of [(3)H]bilirubin di-[(14)C]glucuronide in homozygous Gunn rats, 5-7% of the injected pigment was excreted in bile as BMG. The (14)C:(3)H ratio in the injected BDG was 10% greater than the (14)C:(3)H ratio in BMG excreted in bile. These results indicate that in vivo, dismutation rather than partial hydrolysis, is responsible for BMG formation. Incubation of [(3)H]bilirubin, BDG and a rat liver plasma membrane preparation resulted in formation of BMG (3.3 nmol/min per mg protein) indicating that dismutation is also reversible in vitro.

Binding of 3'-methyl-N,N-dimethyl-4-aminoazobenzene metabolites to rat liver cytosol proteins and ligandin subunits.

Twenty min after i.p. administration of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene in corn oil to rats, 0.73% of administered radioactivity was present in the liver. Only 0.45% of radioactivity present in liver was recovered in the nuclear fraction, whereas 25% was present in the cytosol fraction. Twenty-seven % of cytosolic radioactivity was trichloroacetic acid precipitable, and 2% was immunoprecipitable with monospecific anti-rat liver ligandin immunoglobulin G. After 3 hr of administration, 3.2% of administered radioactivity was present in the liver, 40% of which was in the cytosol. Although 59% of radioactivity present in liver cytosol was trichloroacetic acid precipitable as compared to 27% at 20 min, the radioactivity precipitated by anti-ligandin immunoglobulin G was still 2%. When liver cytosol obtained from rats after 20 min of 3'-[14C]methyl-N,N-dimethyl-4-aminoazobenzene administration was fractionated on a Sephadex G-75 column, three peaks of radioactivity were observed. When cytosol was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was mainly associated with 5 proteins with molecular weights of 88,000, 47,000, 41,000, 31,000, and 22,000. When the immunoprecipitate obtained from cytosol with anti-ligandin immunoglobulin G was subjected to sodium dodecyl sulfate gel electrophoresis and fluorography, radioactivity was exclusively associated with the subunit of ligandin with a molecular weight of 22,000. Approximately 90% of the radioactivity in the immunoprecipitate was covalently associated with this subunit. These studies reveal that 3'-methyl-N,N-dimethyl-4-aminoazobenzene or its metabolites are selectively bound to the subunit of ligandin with a molecular weight of 22,000 and four other cytosol proteins in vivo.

Examination of intramolecular heterogeneity of plasma membrane protein degradation in canine renal tubular epithelial cells and in rat liver.

We have examined the hypothesis that hydrophilic portions of membrane-bound proteins which lie on either side of the phospholipid bilayer may be degraded at a different rate than are the hydrophobic portions of membrane proteins which are within the bilayer. Plasma membrane fractions from cells of the Maden-Darby canine kidney cell line and rat liver were digested with papain and pronase to cleave a mixture of peptides which is enriched in hydrophilic amino acids. It is proposed that these peptides are derived from regions of membrane-bound proteins which lie outside the bilayer. The residual particulate protein is enriched in hydropholic amino acids and presumably contains the portion of membrane-bound proteins which are in direct contact with the bilayer. A double-isotope method was used to assess the relative degradation rates of these two protein fractions. There was no measurable difference in protein degradation rates between the two fractions and the initial plasma membranes. These results suggest that the intramolecular heterogeneity which results from insertion of membrane-bound proteins into a bilayer is not a factor in their degradation.

Delipidation and reactivation of UDPglucuronosyltransferase from rat liver.

UDPglucuronosyltransferase was solubilized by treating Wistar rat liver microsomes with deoxycholate. Chromatography of this preparation on Bio-Gel P-30 resulted in extraction of 92% of phospholipids and complete loss of enzyme activity. UDPglucuronosyltransferase was reactivated by dialysing this delipidated preparation in the presence of lecithin, a mixture of liver microsomal lipids or microsomal preparations from livers of UDPglucuronosyltransferase-deficient Gunn rats. Virtually complete enzyme reactivation was obtained with regard to glucuronidation and glucosidation of bilirubin; however, the inactivation of UDPglucuronosyltransferase with p-nitrophenol as substrate was irreversible. These findings demonstrate that UDPglucuronosyltransferase with bilirubin as substrate is a lipid-requiring enzyme.

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 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.

The role of thiols in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione by rat liver plasma membrane vesicles.

The effect of thiol/disulfide exchange on ATP-dependent S-(2,4-dinitrophenyl)glutathione (GS-DNP) transport was studied in sodium nitrate treated rat liver plasma membrane vesicles. Transport followed Michaelis-Menten kinetics with an apparent Km of 9.6 microM for GS-DNP and 124 microM for ATP. 5,5'-Dithiobis(2-nitrobenzoate) (DTNB) and N-ethylmaleimide (NEM) efficiently inactivated GS-DNP transport activity in a dose- and time-dependent manner. Half-maximal inactivation occurred in 10 min at 40 microM for DTNB and 550 microM for NEM. Inactivation by DTNB was reversed by dithiothreitol. S-(N-Ethyl)maleimyl glutathione and/or ATP-Mg2+, but neither S-(N-ethyl)maleimyl cysteinylglycine nor oxidized glutathione could protect transport activity from inactivation by NEM or cystamine. These results suggest that reactive thiols are located near the active site of the transporter and that S-alkyl and the gamma-glutamyl residues of glutathione are important for protection. Biological disulfides which were tested included cystine, oxidized glutathione, oxidized Coenzyme-A, oxidized lipoic acid, and oxidized lipoamide; cystamine was the most potent reversible inactivator. Molecular oxygen also inactivated transport activity, which was recovered on addition of dithiothreitol, suggesting intramolecular disulfide formation by vicinal thiols. We interpret these results to indicate that the ATP-dependent GS-DNP transporter contains two or more thiols which are necessary for the maintenance of transport activity. The reversible inactivation of the activity by biological disulfides suggests that the transporter may be regulated by thiol/disulfide exchange in vivo.

Circular dichroism analysis of the secondary structure of Z protein and its complexes with bilirubin and other organic anions.

Circular dichroism (CD) methods were employed to study the conformation of Z protein and characterize its complexes with bilirubin and other organic anions. Z protein-bilirubin complexes exhibited a spectrum with overlapping ellipticity bands of opposite sign in the bilirubin absorption region. These results were compared with those obtained with ligandin, the other major organic anion binding protein of liver. Secondary structural differences between the two proteins were easily demonstrated since ligandin is predominantly an alpha-helical protein and Z features mainly beta-structure. Furthermore, the optical activity pattern generated by bilirubin binding to Z was virtually a mirror image of that of the ligandin bilirubin system. CD experiments were designed to study the direct transfer of bilirubin between Z protein and ligandin, and it was shown that both proteins have almost equal affinities for bilirubin. The bilirubin on Z was readily displaced by oleic acid and displaced to a lesser extent by sulfobromophthalein,

Bilirubin and biliverdin binding to rat Y protein (ligandin).

The binding of bilirubin to poly(L-lysine) produces an optically active complex at pH 10.1. Circular dichroism spectra of these complexes are distinguishable from those generated by binding of bilirubin to the high affinity sites on albumin. Comparison of the circular dichroism spectra of bilirubin bound to the hepatic protein ligandin with those of bilirubin complexed with albumin or polylysine indicates that binding of bilirubin to ligandin occurs at two types of sites. These are distinguishable on the basis of their spectral properties, one resembling the high affinity site of bovine serum albumin and the other resembling polylysine. Complexes of biliverdin with albumin and ligandin bear similarities to the bilirubin-protein complexes. The native protein itself has an ordered structure which consists of 41% alpha-helix and is not altered by the binding of bilirubin.