Biomedical subjects
Publications and source records attributed to K Kamisaka.
Preparation of Z protein from human liver is described. Z protein consists of 2 forms which have different isoelectric points, pI 5.8 and pI 8.7 respectively. The acidic Z protein has a molecular weight of about 11,000 and has binding affinity for BSP using gel filtration.
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Sephadex G-100 chromatography of rat liver supernatant after addition of [125I]T3 revealed four peaks of protein-bound radioactivity in the void volume, albumin, ligandin, and Z-containing regions, respectively. The peaks were identified by cochromatography of BSP and [125I]T3 and immonodiffusion with antiratligandin IgG and antirat Z IgG. Binding of [125I]T4 to rat liver supernatant occurred in void volume, albumin, and Z regions only. Studies in vivo reveal a pattern of [125I]T3 binding to rat liver supernatant fractions quantitatively different from that observed in vitro. [125I]T4 binding to liver supernatant fractions in vivo occurred in all four peaks. BSP or bilirubin added to liver supernatant decreased T3 and T4 binding by each fraction. Flavaspidic acid inhibited binding of T3 and T4 to albumin, ligandin, and Z protein. Phenobarbital pretreatment of rats increased binding of T3 by ligandin and of T4 by albumin-containing fractions. Circular dichroism studies with purified rat liver ligandin suggest that T3 and T4 bind competitively to the same site as does bilirubin; the association constants of T3 and T4 for ligandin are 10(6) and 10(5) M-1, respectively. T4 was bound only by purified ligandin and not by ligandin in liver supernatant. To determine whether unconjugated bilirubin interferes with hepatic uptake of T3, [125I]T3 was administered to icteric homozygous and phenotypically normal heterozygous Gunn rats. Hepatic uptake and supernatant binding [125I]T3 were significantly reduced in homozygous Gunn rats. Hepatic uptake of [125I]T3 was also reduced in vivo by infusion of BSP with or without flavaspidic acid. BSP infusion abolished [125I]T3 binding to ligandin; BSP and flavaspidic acid abolished binding to ligandin and Z. These observations suggest that ligandin and Z protein are thyroid hormone binding proteins in rat liver cytosol and may influence the net flux of iodothyronies from plasma into the liver.
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The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate-gel electrophoresis. Transferases alpha, beta and epsilon each appear as a single band on gel electrofocusing; transferases gamma and delta are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate.
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,
Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.
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.
Ligandin, an abundant cytoplasmic binding protein of bilirubin and other ligands in liver cells, completely prevented the inhibitory effect of bilirubin on respiration and oxidative phosphorylation by isolated rat liver mitochondria. At equimolar concentrations of bilirubin and ligandin or human serum albumin, mitochondrial respiration was fully restored to control values. At greater ratios of bilirubin and ligandin or human serum albumin, the latter had a stronger protective effect. These studies suggest that ligandin may have a physiologic role in protecting mitochondrial systems against bilirubin toxicity.
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